1000. Serotype 3 pneumococci evade activation of the classical complement pathway

Background Complement classical pathway (CCP) activation is the major mechanism leading to opsonophagocytic pneumococcal killing. Following immunization with 13-valent pneumococcal conjugate vaccine (PCV13), opsonophagocytic titers are lowest against serotype 3 among the 13 vaccine serotypes. Post licensure surveillance indicated early declines in serotype 3 invasive pneumococcal disease (IPD) were not sustained over time Methods Using flow cytometry, we measured C3 and C4 deposition on serotype 3 strains from children with IPD or nasopharyngeal [NP] carriage, and analyzed by clade. C4 deposition is an indicator of CCP, while C3 deposition is common to all complement pathways. We measured C3/C4 deposition on serotype 3 pneumococcal strains incubated with antibody depleted complement alone or with complement and the following antibodies: mouse monoclonal anti-capsular IgG or IgM, rabbit polyclonal serotype 3 antisera (IgG + IgM) [RPS3A] and RPS3A combined with anti-rabbit IgM, which blocks IgM function, leaving only polyclonal IgG Results Serotype 3 strains demonstrated high variability in C3 binding when incubated with complement alone. RPS3A (containing both IgM+IgG) and monoclonal IgM activated CCP in all strains. Anti- serotype 3 monoclonal IgG and polyclonal IgG demonstrated absent or limited CCP activation; but activated alternative pathway in some strains. When analyzing complement deposition by clade, a lower proportion of clade II NP serotype 3 strains bound C3 when incubated with complement or monoclonal IgG, compared to clade Ia NP strains. Differences between clade Ia and II IPD strains were not apparent. Conclusion Serotype 3 strains did not demonstrate activation of the CCP in the presence IgG and varied in C3 deposition. Pneumococcal strains that evade CCP activation may be less sensitive to opsonophagocytosis. Our findings suggest a mechanism by which serotype 3 carriage and disease may persist despite immunization with conjugate vaccine containing serotype 3 polysaccharide. Disclosures Rotem Lapidot, MD MSCI, Pfizer (Consultant, Grant/Research Support, Advisor or Review Panel member) Mario Ramirez, PhD, GlaxoSmithKline (Advisor or Review Panel member)Merck Sharp & Dohme (Advisor or Review Panel member)Pfizer (Speaker’s Bureau) Ingrid L. Scully, PhD, Pfizer (Employee, Shareholder) Bradford D. Gessner, MD, MPH, Pfizer Inc. (Employee) stephen pelton, MD, Merck Vaccines (Advisor or Review Panel member, Research Grant or Support)Pfizer, Inc. (Consultant, Advisor or Review Panel member, Research Grant or Support)Sanofi pasteur (Advisor or Review Panel member, Research Grant or Support, DSMB)Seqirus (Consultant)

Enhancement of Binding Affinity of Anti-Hapten Polyclonal IgG Recognizing Mitragynine using Affinity Purification

Antibodies are glycoproteins found in peritoneal fluid, serum, and blood. The antibody-based assay has been used for broad applications such as immunodiagnostic and other biomedical applications. Depending on the intended application, a highly purified polyclonal antibody could be used as an alternative. Purification of antibodies from anti-sera has been proven as one of the methods to enhance the binding affinity of antibodies towards its antigen. We report herein the enhancement of the binding affinity of anti-hapten polyclonal IgG recognizing mitragynine using affinity purification. Serum from the terminal bleed of New Zealand White (NZW) rabbits immunized with mitragynine conjugated with cationized– bovine serum albumin at methyl ester (C22-MG-cBSA), or aromatic ether modification (C9-MG-cBSA) were subjected to HiTrap Protein G affinity purification using fast protein liquid chromatography (FPLC). The elution peak from chromatography fractions was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Here, we report the binding of polyclonal antibodies produced from inoculation of either C22-MG-cBSA or C9-MG-cBSA immunogens of which mitragynine-ovalbumin (MG-OVA) was used as coating antigen in the ELISA assay. Non purified anti-sera from C22-MG-cBSA-inoculated rabbits showed higher titer than C9-MG-cBSA at 1/128 000 and 1/32 000 dilutions, respectively. The affinity of purified poly-IgGs from rabbits immunized with C22-MG-cBSA showed a mean Kd value of 7.965 × 10-6 μM, which was lower than those immunized with C9-MG-cBSA at mean Kd of 1.390 × 10-4 μM. In addition, the purified poly- IgGs showed higher binding towards MG-OVA than non-purified anti-sera at comparable protein concentrations. These results indicated that the higher binding affinity of purified polyclonal IgG is due to the reduced competition among polyclonal antibodies with non- IgG proteins that co-existed in the non-purified anti-sera after the affinity purification.

Experimental modeling of behavioral disorders accompanying hashimoto’s thyroiditis by means of specific immunoglobulins

Among the manifestations of Hashimotos autoimmune thyroiditis, there are various psychoneurological disorders. For more than a century, it has been known about psycho-neurological disorders associated with hypothyroidism, but along with that, there are also mental disorders in patients with thyropathies in euthyroid state. In 1966, Hashimotos encephalopathy was described, the pathogenesis and clear differential diagnostic criteria of which have not yet been determined. This article describes an experimental study in laboratory mice with intracisternal stereotaxic injection of IgG isolated from patients with autoimmune thyroiditis and comorbid depression or schizophrenia. A control group included animals receiving polyclonal IgG from healthy donors. Then behavioral tests were carried out, which revealed the characteristics and changes in behavior in the operated animals. Thus, animals that received immunoglobulins from patients with autoimmune thyroiditis and depression were less active in relation to the development of risk behavior. Porsolts tests on the 4th and 15th days after surgery showed that, regardless of the kind of the injected solutions, there was a change in the temporal relationships between the behavior patterns. In mice received IgG from patients with autoimmune thyroiditis and schizophrenia during the delayed Porsolt test, the ratio of the forms of motor activity shifted towards passive swimming. The mice received IgG from healthy donors did not demonstrate this change.

Polyclonal alpaca antibodies protect against hantavirus pulmonary syndrome in a lethal Syrian hamster model

The use of antibody-based therapies for the treatment of high consequence viral pathogens has gained interest over the last fifteen years. Here, we sought to evaluate the use of unique camelid-based IgG antibodies to prevent lethal hantavirus pulmonary syndrome (HPS) in Syrian hamsters. Using purified, polyclonal IgG antibodies generated in DNA-immunized alpacas, we demonstrate that post-exposure treatments reduced viral burdens and organ-specific pathology associated with lethal HPS. Antibody treated animals did not exhibit signs of disease and were completely protected. The unique structures and properties, particularly the reduced size, distinct paratope formation and increased solubility of camelid antibodies, in combination with this study support further pre-clinical evaluation of heavy-chain only antibodies for treatment of severe respiratory diseases, including HPS.

Protection of human ACE2 transgenic Syrian hamsters from SARS CoV-2 variants by human polyclonal IgG from hyper-immunized transchromosomic bovines

Pandemic SARS CoV-2 has been undergoing rapid evolution during spread throughout the world resulting in the emergence of many Spike protein variants, some of which appear to either evade antibody neutralization, transmit more efficiently, or potentially exhibit increased virulence. This raises significant concerns regarding the long-term efficacy of protection elicited after primary infection and/or from vaccines derived from single virus Spike (S) genotypes, as well as the efficacy of anti-S monoclonal antibody based therapeutics. Here, we used fully human polyclonal human IgG (SAB-185), derived from hyperimmunization of transchromosomic bovines with DNA plasmids encoding the SARS-CoV-2 Wa-1 strain S protein or purified ectodomain of S protein, to examine the neutralizing capacity of SAB-185 in vitro and the protective efficacy of passive SAB-185 antibody (Ab) transfer in vivo. The Ab preparation was tested for neutralization against five variant SARS-CoV-2 strains: Munich (Spike D614G), UK (B.1.1.7), Brazil (P.1) and SA (B.1.3.5) variants, and a variant isolated from a chronically infected immunocompromised patient (Spike del144-146). For the in vivo studies, we used a new human ACE2 (hACE2) transgenic Syrian hamster model that exhibits lethality after SARS-Cov-2 challenge and the Munich, UK, SA and del144-146 variants. SAB-185 neutralized each of the SARS-CoV-2 strains equivalently on Vero E6 cells, however, a control convalescent human serum sample was less effective at neutralizing the SA variant. In the hamster model, prophylactic SAB-185 treatment protected the hamsters from fatal disease and minimized clinical signs of infection. These results suggest that SAB-185 may be an effective treatment for patients infected with SARS CoV-2 variants.

Purification of polyclonal Immunoglobulin G from human serum using LigaGuardTM and LigaTrapTM adsorbents

This study presents the chromatographic purification of immunoglobulin G (IgG) from human plasma using a two-column process integrating the peptide-based adsorbents LigaGuardTM, which captures non-Ig plasma proteins in flow-through mode, and LigaTrapTM, which isolates IgG in bind-and-elute. Buffer composition and column loading were optimized for both adsorbents. Two process configurations were evaluated. In the first design, plasma was fed to a LigaGuardTM column to capture plasma proteins, the effluent was loaded on the LigaTrapTM column, and the bound IgG was eluted with 63.8% global recovery and 99.7% purity; in comparison, Protein G agarose afforded ~67% recovery and 97.2% purity. In the alternative design, the LigaGuardTM column was utilized to polish the LigaTrapTM elution stream, affording 82.3% global recovery and 98.8% purity. Collectively, these results demonstrate the potential of a fully chromatographic process for purifying polyclonal IgG from plasma feedstocks.

Influence of Caudovirales Phages on Humoral Immunity in Mice

Bacteriophages are promising antibacterial agents. Although they have been recognized as bacterial viruses and are considered to be non-interacting with eukaryotic cells, there is growing evidence that phages may have a significant impact on the immune system via interactions with macrophages, neutrophils, and T-cell polarization. In this study, the influence of phages of podovirus, siphovirus, and myovirus morphotypes on humoral immunity of CD-1 mice was investigated. In addition, tissue distribution of the phages was tested in these mice. No common patterns were found either in the distribution of phages in mice or in changes in the levels of cytokines in the sera of mice once injected with phages. Importantly, pre-existing IgM-class antibodies directed against capsid proteins of phages with myovirus and siphovirus morphotypes were identified in mice before immunization. After triple immunization of CD1-mice with phages without any adjuvant, levels of anti-phage serum polyclonal IgG antibodies increased. Immunogenic phage proteins recognized by IgM and/or IgG antibodies were identified using Western blot analysis and mass spectrometry. In addition, mice serum collected after immunization demonstrated neutralizing properties, leading to a substantial decrease in infectivity of investigated phages with myovirus and siphovirus morphotypes. Moreover, serum samples collected before administration of these phages exhibited some ability to reduce the phage infectivity. Furthermore, Proteus phage PM16 with podovirus morphotype did not elicit IgM or IgG antibodies in immunized mice, and no neutralizing activities against PM16 were revealed in mouse serum samples before and after immunization.

Production and purification of polyclonal anti-chicken IgY immunoglobulins in Goats and conjugation with Horseradish Peroxidase

Antibodies are very important components in medical diagnostics and various biochemical assays for diagnosis of the diseases. IgY is an immunoglobulin largely present in Avians. Anti-chicken IgY immunoglobulins conjugated with gold nanoparticles are used to diagnose the chicken infectious diseases by lateral flow assay (LFA) and enzyme linked immunosorbent assay (ELISA) immunodiagnostic tests. The main aim of this study is the production of antichicken IgY antibodies in goats, purification of antiantibody and conjugation with horse radish peroxidase (HRP) enzyme. The antibody was purified using protein G sepharose affinity column and the purity of the antibody was confirmed by SDS-PAGE analysis and by spectrophotometer. The purified anti IgY antibody was also evaluated by ELISA. The optimum dilution of prepared HRP conjugated anti IgY was 1:9600. The sensitivity and specificity of the antibody were evaluated and have no cross reactivity with other IgG antibodies. This study showed that protein G affinity purification could be best useful method for purification of polyclonal IgG antibodies.

DNA hydrolysing IgG catalytic antibodies: an emerging link between psychoses and autoimmunity

It is not uncommon to observe autoimmune comorbidities in a significant subset of patients with psychotic disorders, namely schizophrenia (SCZ) and bipolar disorder (BPD). To understand the autoimmune basis, the DNA abyzme activity mediated by serum polyclonal IgG Abs were examined in psychoses patients, quantitatively, by an in-house optimized DNase assay. A similar activity exhibited by IgG Abs from neuropsychiatric-systemic lupus erythematosus (NP-SLE) patients was used as a comparator. Our data revealed that the IgG DNase activity of SCZ was close to that of NP-SLE and it was twofold higher than the healthy controls. Interestingly, the association between DNase activity with PANSS (positive, general and total scores) and MADRS were noted in a subgroup of SCZ and BPD patients, respectively. In our study group, the levels of IL-6 and total IgG in BPD patients were higher than SCZ and healthy controls, indicating a relatively inflammatory nature in BPD, while autoimmune comorbidity was mainly observed in SCZ patients.

Daratumumab in combination with proteasome inhibitors, rapidly decreases polyclonal immunoglobulins and increases infection risk among relapsed multiple myeloma patients: a single center retrospective study

Background: Daratumumab (Dara) is generally well tolerated, but is associated with increased risk of infection. Methods: We investigated hypogammaglobinemia occurrence in different Dara-based regimens. Multiple myeloma (MM) patients were treated with ⩾2 cycles of Dara-based therapy during 2016–2020, mainly for relapsed/refractory disease. Data on patient characteristics, treatment regimens, polyclonal IgG (poly-IgG) and uninvolved free light chain (Un-FLC) levels during treatment, as well as predictors for hypogammaglobinemia and predictors for infections, were evaluated retrospectively. Results: A total of 84 patients, median age 67.2 years, were included. Dara, mainly as ⩾2 line therapy (88.1%, n = 74), was combined with immunomodulating drugs (IMiDs) (53%), proteasome inhibitors (PIs) (15%), IMiDs-PIs (11%), or dexamethasone only (21%). Median treatment duration was 13 months. Median Poly-IgG levels at 0, 2, and 4 months were 7.1 g/l, 4.5 g/l, and 4 g/l, respectively, and remained low throughout treatment. Lower poly-IgG pre-Dara ( p = 0.001) and Dara-PIs (±IMiDs) regimen were associated with lower poly-IgG levels at 4 months ( p = 0.03). Only patients treated with Dara monotherapy had partial immune reconstitution, reflected by resumption of IgM levels. Most (85%) patients developed ⩾1 infections, mostly grade 1–2 respiratory (76%). A lower poly-IgG level post Dara (RR = 1.137 p = 0.026) predicted increased risk of any infection. Intravenous immunoglobulin (IVIG) was associated with a significant decrease in all infections. Conclusion: Relapsed MM patients treated with Dara, often experience persistent hypogammaglobinemia, irrespective of responsiveness to treatment. Infections, especially respiratory, are frequent and apparently related to low Poly-IgG levels. IVIG should be considered for reducing infections in these patients.