Spike-Dependent Opsonization Indicates Both Dose-Dependent Inhibition of Phagocytosis and That Non-Neutralizing Antibodies Can Confer Protection to SARS-CoV-2

Spike-specific antibodies are central to effective COVID19 immunity. Research efforts have focused on antibodies that neutralize the ACE2-Spike interaction but not on non-neutralizing antibodies. Antibody-dependent phagocytosis is an immune mechanism enhanced by opsonization, where typically, more bound antibodies trigger a stronger phagocyte response. Here, we show that Spike-specific antibodies, dependent on concentration, can either enhance or reduce Spike-bead phagocytosis by monocytes independently of the antibody neutralization potential. Surprisingly, we find that both convalescent patient plasma and patient-derived monoclonal antibodies lead to maximum opsonization already at low levels of bound antibodies and is reduced as antibody binding to Spike protein increases. Moreover, we show that this Spike-dependent modulation of opsonization correlate with the outcome in an experimental SARS-CoV-2 infection model. These results suggest that the levels of anti-Spike antibodies could influence monocyte-mediated immune functions and propose that non-neutralizing antibodies could confer protection to SARS-CoV-2 infection by mediating phagocytosis.

Performance of a flow cytometry-based immunoassay for detection of antibodies binding to SARS-CoV-2 spike protein

The performance of a laboratory-developed IgG/IgA flow cytometry-based immunoassay (FCI) using Jurkat T cells stably expressing full-length native S protein was compared against Elecsys electrochemiluminiscent (ECLIA) Anti-SARS-CoV-2 S (Roche Diagnostics, Pleasanton, CA, USA), and Liaison SARS-CoV-2 TrimericS IgG chemiluminiscent assay (CLIA) (Diasorin S.p.a, Saluggia, IT) for detection of SARS-CoV-2-specific antibodies. A total of 225 serum/plasma specimens from 120 acute or convalescent COVID-19 individuals were included. Overall, IgG/IgA-FCI yielded the highest number of positives (n = 179), followed by IgA-FCI (n = 177), Roche ECLIA (n = 175), IgG-FCI (n = 172) and Diasorin CLIA (n = 154). For sera collected early after the onset of symptoms (within 15 days) IgG/IgA-FCI also returned the highest number of positive results (52/72; 72.2%). Positive percent agreement between FCI and compared immunoassays was highest for Roche ECLIA, ranging from 96.1 (IgG/IgA-FCI) to 97.7% (IgG-FCI), whereas negative percent agreement was higher between FCI and Diasosin CLIA, regardless of antibody isotype. The data suggest that FCI may outperform Roche ECLIA and Diasorin CLIA in terms of clinical sensitivity for serological diagnosis of SARS-CoV-2 infection.

Anti-PEG antibodies boosted in humans by SARS-CoV-2 lipid nanoparticle mRNA vaccine

Humans commonly have low level antibodies to poly(ethylene) glycol (PEG) due to environmental exposure. Lipid nanoparticle mRNA vaccines for SARS-CoV-2 contain small amounts of PEG but it is not known if PEG antibodies are enhanced by vaccination and if there are any consequences. We studied plasma from 55 people receiving the Comirnaty (Pfizer-BioNTech) mRNA vaccine for PEG-specific antibodies. Anti-PEG IgG was commonly detected prior to vaccination and was boosted a mean of 1.78-fold (range 0.68 to 16.6) by vaccination. Anti-PEG IgM increased 2.64-fold (0.76 to 12.84) following vaccination. PEG antibodies did not impact the neutralizing antibody response to vaccination. Pre-existing levels of anti-PEG IgM correlated with increased reactogenicity. A rise in PEG antibodies following vaccination was associated with an increase in the association of PEG-based nanoparticles to blood immune cells ex vivo. We conclude that low level PEG-specific antibodies can be modestly boosted by a lipid nanoparticle mRNA-vaccine and that PEG-specific antibodies are associated with higher reactogenicity. The longer-term clinical impact of the increase in PEG-specific antibodies induced by lipid nanoparticle mRNA-vaccines should be monitored.

Diminished antibody response against SARS-CoV-2 Omicron variant after third dose of mRNA vaccine in kidney transplant recipients

Background: Available SARS-CoV-2 vaccines have reduced efficacy against the Omicron variant in immunocompetent individuals. Kidney transplant recipients (KTRs) have diminished antiviral responses to wild-type SARS-CoV-2 after vaccination, and data on antiviral responses to SARS-CoV-2 variants, including the Omicron variant, are limited. Methods: We conducted a prospective, multi-center cohort study of 51 adult KTRs who received three doses of BNT162b2 or mRNA-1273. Blood and urine samples were collected before and four weeks after the third vaccine dose. The primary outcome was anti-viral antibody responses against wild-type and variants of SARS-CoV-2. Secondary objectives included occurrence of breakthrough SARS-CoV-2 infection and non-invasive monitoring for rejection using serum creatinine, proteinuria, donor-derived cell-free DNA and donor-specific antibodies. Sera from pre-pandemic healthy controls and KTRs were used for comparison. Results: 67% of KTRs developed anti-wild-type spike antibodies after the third vaccine dose, similar to the Alpha (51%) and Beta (53%) variants, but higher than the Gamma (39%) and Delta (25%) variants. No KTRs had neutralizing responses to the Omicron variant before the third vaccine dose. After the third dose, fewer KTRs had neutralizing responses to the Omicron variant (12%) compared to wild-type (61%) and Delta (59%) variants. Three patients (6%) developed breakthrough SARS-CoV-2 infection at a median of 89 days. No KTRs developed allograft injury, de novo donor-specific antibodies or allograft rejection. Conclusion: In KTRs, a third dose of mRNA vaccines increases antibody responses against wild-type and variants of SARS-CoV-2, while neutralizing responses to the Omicron variant remain markedly reduced.

Heterologous vaccination with inactivated and mRNA vaccines increases B and T cell responses to SARS-CoV-2

Background There has been an unprecedented global effort to produce safe and effective vaccines against SARS-CoV-2. However, production challenges, supply shortages and unequal global reach, together with an increased number of breakthrough infections due to waning of immunity and the emergence of new variants of concern (VOC), have prolonged the pandemic. To boost the immune response, several heterologous vaccination regimes have been tested and have shown increased antibody responses compared to homologous vaccination. Here we evaluated the effect of mRNA vaccine booster on immunogenicity in individuals who had been vaccinated with two doses of inactivated vaccines. Methods The levels of specific antibodies against the receptor-binding domain (RBD) of the spike protein from wild-type virus and the Beta, Delta and Omicron variants were measured in healthy individuals who had received two doses of homologous inactivated (BBIBP-CorV or CoronoVac) or mRNA (BNT162b2 or mRNA-1273) vaccines, and in donors who were given an mRNA vaccine boost after two doses of either vaccine. Pre-vaccinated healthy donors, or individuals who had been infected and subsequently received the mRNA vaccine were also included as controls. In addition, specific memory B and T cell responses were measured in a subset of samples. Results A booster dose of an mRNA vaccine significantly increased the specific antibody response to the wild-type and VOCs including Omicron (by 14-fold), in individuals who had previously received two doses of inactivated vaccines. The levels of specific antibodies in the heterologous vaccination group were similar to those in individuals receiving a third dose of homologous mRNA vaccines or boosted with mRNA vaccine after natural infection. Furthermore, this heterologous vaccination regime significantly improved the specific memory B and T cell responses. Conclusions Heterologous prime-boost immunization with inactivated vaccine followed by an mRNA vaccine boost markedly increased the levels of specific antibodies and B and T cell responses and may thus increase protection against emerging SARS-CoV-2 variants including Omicron.

Induction of Functional Specific Antibodies, IgG-Secreting Plasmablasts and Memory B Cells Following BCG Vaccination

Tuberculosis (TB) is a major global health problem and the only currently-licensed vaccine, BCG, is inadequate. Many TB vaccine candidates are designed to be given as a boost to BCG; an understanding of the BCG-induced immune response is therefore critical, and the opportunity to relate this to circumstances where BCG does confer protection may direct the design of more efficacious vaccines. While the T cell response to BCG vaccination has been well-characterized, there is a paucity of literature on the humoral response. We demonstrate BCG vaccine-mediated induction of specific antibodies in different human populations and macaque species which represent important preclinical models for TB vaccine development. We observe a strong correlation between antibody titers in serum versus plasma with modestly higher titers in serum. We also report for the first time the rapid and transient induction of antibody-secreting plasmablasts following BCG vaccination, together with a robust and durable memory B cell response in humans. Finally, we demonstrate a functional role for BCG vaccine-induced specific antibodies in opsonizing mycobacteria and enhancing macrophage phagocytosis in vitro, which may contribute to the BCG vaccine-mediated control of mycobacterial growth observed. Taken together, our findings indicate that the humoral immune response in the context of BCG vaccination merits further attention to determine whether TB vaccine candidates could benefit from the induction of humoral as well as cellular immunity.

Immunobiological Properties of Antigenic Preparations Streptococcus pneumoniae and their Mixtures

Relevance. Type-specific immunity does not protect against infection with other pneumococcal serotypes. The phenomenon of the change of serotypes dominating the population of Streptococcus pneumoniae is known, in part due to the intensive recombination process and the phenomenon of «capsule switching». Therefore, the development of a serotype-independent pneumococcal vaccine is an important global public health priority. Ams. Investigation of immunobiological properties of candidate components of a future vaccine with serotype-independent activity. Materials and methods. For immunization of mice, preparations of the capsular polysaccharide of pneumococcus serotype 3 (CPS) were used; protein-containing fraction (PCF) obtained from an aqueous extract of S. pneumoniae 6B cells; recombinant pneumolysin (Ply); mixtures of drugs (CPS + Plу; CPS + PCF; PCF + Plу); conjugate vaccine Prevnar 13 (manufactured by PFIZER Inc. USA). Mice were immunized intraperitoneally, 2 times with an interval of 14 days. Intact mice were used as a control group. To assess the humoral immune IgG response, the method of solid-phase ELISA was used. Phagocytic activity was studied at 7, 14, 21 and 28 days after the second immunization. The cytokine level was determined in the blood sera of mice after the second immunization 2, 4, 8, and 24 hours later on a NovoCyte flow cytometer (ACEA Biosciences, USA) using the MACSPIex CytoKine 10 Kit mouse (Miltenyi Biotec Inc., USA) according to the manufacturer's instructions. Results. Immunization of mice with Ply as well as mixtures with CPS and PCF caused a significant increase in the level of antibodies to Ply. It was found that there was no apparent decrease in the level of antigen-specific antibodies when antigens were administered in combination with others. Pneumolysin, used alone or in combination with PCF and CPS, induces the production of antiinflammatory cytokines IL-4, IL-10, and IL-5 detected throughout the study. This is confirmed by a study of the opsonophagocytic activity of neutrophils from immunized CPS + Ply, Ply + PCF and Ply mice; a significant increase in the number of eosinophils is observed in their blood due to the stimulation of their production of IL-5. Conclusions. As a result of the studies, it was shown that Ply, used alone or in combination with CPS and PCF, has the highest immunogenicity: it stimulates a significant increase in the level of specific antibodies, stimulates Th-2, and induces the production of anti-inflammatory cytokines.

Immune responses to inactivated and vector-based vaccines in individuals previously infected with SARS-CoV-2

Immunity wanes in individuals previously infected with SARS-CoV-2, and vaccinating those individuals may help reduce reinfection. Herein, reactogenicity and immunogenicity following vaccination with inactivated (CoronaVac) and vector-based (ChAdOx1-S, AZD1222) vaccines were examined in previously infected individuals. Immune response was also compared between short and long intervals between first date of detection and vaccination. Adverse events were mild but were higher with AZD1222 than with CoronaVac. Baseline IgG-specific antibodies and neutralizing activity were significantly higher with shorter than longer intervals. With a single-dose vaccine, IgG and IgA-specific binding antibodies, neutralizing activity, and total interferon-gamma response peaked at 14 days. Immune response was significantly higher in recovered individuals than in infection-naïve individuals. Antibody response was greater with longer than shorter intervals. AZD1222 induced higher antibody and T cell responses than those of CoronaVac. Thus, to achieve immunity, individuals with prior SARS-CoV-2 exposure may require only a single dose of AZD1222 or two doses of CoronaVac to achieve the immune response. These findings supported vaccine strategies in previously infected individuals.

The gray area between operational tolerance and overt rejection in kidney transplantation

Operational tolerance in kidney transplantation is characterized by stable serum creatinine < 1.7 mg/dL and proteinuria < 1 g/day in the absence of immunosuppression or immunodeficiency for over one year. However, simultaneous donor specific antibodies are common and serum creatinine is a poor surrogate of early lesions. Consequently, subclinical rejections will meet operational tolerance criteria if serum creatinine remains stable. We report a patient with operational tolerance criteria followed by biopsy-proven chronic active antibody mediated rejection, discussing the intricate challenges of immunosuppression management.

Cruzipain Sulfotopes-Specific Antibodies Generate Cardiac Tissue Abnormalities and Favor Trypanosoma cruzi Infection in the BALB/c Mice Model of Experimental Chagas Disease

Trypanosoma cruzi cruzipain (Cz) bears a C-terminal domain (C-T) that contains sulfated epitopes “sulfotopes” (GlcNAc6S) on its unique N-glycosylation site. The effects of in vivo exposure to GlcNAc6S on heart tissue ultrastructure, immune responses, and along the outcome of infection by T. cruzi, were evaluated in a murine experimental model, BALB/c, using three independent strategies. First, mice were pre-exposed to C-T by immunization. C-T-immunized mice (C-TIM) showed IgG2a/IgG1 &lt;1, induced the production of cytokines from Th2, Th17, and Th1 profiles with respect to those of dC-TIM, which only induced IL-10 respect to the control mice. Surprisingly, after sublethal challenge, both C-TIM and dC-TIM showed significantly higher parasitemia and mortality than the control group. Second, mice exposed to BSA-GlcNAc6S as immunogen (BSA-GlcNAc6SIM) showed: severe ultrastructural cardiac alterations while BSA-GlcNAcIM conserved the regular tissue architecture with slight myofibril changes; a strong highly specific humoral-immune-response reproducing the IgG-isotype-profile obtained with C-TIM; and a significant memory-T-cell-response demonstrating sulfotope-immunodominance with respect to BSA-GlcNAcIM. After sublethal challenge, BSA-GlcNAc6SIM showed exacerbated parasitemias, despite elevated IFN-γ levels were registered. In both cases, the abrogation of ultrastructural alterations when using desulfated immunogens supported the direct involvement of sulfotopes and/or indirect effect through their specific antibodies, in the induction of tissue damage. Finally, a third strategy using a passive transference of sulfotope-specific antibodies (IgG-GlcNAc6S) showed the detrimental activity of IgG-GlcNAc6S on mice cardiac tissue, and mice treated with IgG-GlcNAc6S after a sublethal dose of T. cruzi, surprisingly reached higher parasitemias than control groups. These findings confirmed the indirect role of the sulfotopes, via their IgG-GlcNAc6S, both in the immunopathogenicity as well as favoring T. cruzi infection.