July 18, 2024

A competitive hemagglutination inhibition assay for dissecting functional antibody activity to influenza virus

  • Influenza remains one of the most contagious infectious diseases. Approximately, 25-50 million people suffer from influenza-like illness in the United States annually, leading to almost 1 million hospitalizations. Globally, the World Health Organization (WHO) estimates 250,000-500,000 mortalities associated with secondary respiratory complications due to influenza virus infection every year. Currently, seasonal vaccination represents the best countermeasure to prevent influenza virus spread and transmission in the general population. However, presently licensed influenza vaccines are about 60% effective on average, and their effectiveness varies from season to season and among age groups, as well as between different influenza subtypes within a single season.
  • The hemagglutination inhibition (HAI) assay represents the gold standard method for measuring the functional antibody response elicited following standard-of-care vaccination, along with evaluating the efficacy of under-development influenza vaccines in both animal models and clinical trial settings. However, using the classical HAI approach it is not possible to dissect the complexities of variable epitope recognition within a polyclonal antibody response. In this communication, we describe a straightforward competitive HAI-based method using a combination of influenza virus and recombinant hemagglutinin (HA) proteins to dissect the HAI functional activity of HA-specific antibody populations in a single assay format. Importance: The hemagglutination inhibition (HAI) assay is a well-established and reproducible method that quantifies functional antibody activity against influenza viruses and in particular the capability of an antibody formulation to inhibit the binding of HA to sialic acid.
  • However, the HAI assay does not provide with full insights on the breadth and epitope recognition of the antibody formulation, especially in the context of polyclonal sera where multiple antibody specificities contribute to the overall observed functional activity. In this report we introduce the use of Y98F point-mutated recombinant HA (HAΔSA) proteins, which lack sialic acid binding activity, in the context of the HAI assay as a mean to absorb out certain HA-directed (i.e., strain-specific or cross-reactive) antibody populations. This modification to the classical HAI assay, referred to as the competitive HAI assay, represents a new tool to dissect the magnitude and breadth of polyclonal antibodies elicited through vaccination or natural infection

Influence of adjuvants on the amount, specificity and functional activity of antibody response to human influenza vaccine in mice

It’s been almost a century since immunologists started using adjuvants as tools to develop more effective vaccines. Despite the rising number of adjuvanted vaccines in the last decades, we still lack knowledge of the adjuvants’ effects on antibody response. This study was aimed to test the effect of immunizing mice with the human Inactivated Influenza vaccine (IIV), either alone or combined with different widely used adjuvants on the specific antibody response induced. Differential levels of IgM and IgG subclasses were found with the different adjuvants tested. Higher levels of antibodies did not always correspond with a higher efficacy to interfere with the virus infectivity. Differences in neutralization properties are possibly mediated by the specificity of the repertoire of antibodies induced.
The repertoire was studied using a phage display 7-mer peptide library to screen for epitopes/mimotopes recognized by serum pools from vaccinated mice. The selected phage clones included peptides that corresponded to conformational mimotopes since they have no homology with lineal sequences of the Influenza strains’ proteins. Five peptides were identified as recognized by sera from mice immunized with the IIV vaccine alone, including peptides from the hemagglutinin stalk domain, and by sera from mice immunized with the vaccine plus the different adjuvants employed. Adjuvants elicited a more diverse repertoire of epitope-recognizing antibodies that recognized epitopes of the HA recombinant globular head. Mimotopes were theoretically located at the neutralizing antigenic sites of the globular head of Influenza A H1N1pdm09, Influenza A H3N2, and Influenza B hemagglutinin. This study illustrates how different adjuvants can modify the extent and quality of humoral immunity against the IIV vaccine and the effectiveness of vaccination.

Partial Degradation of Recombinant Antibody Functional Activity During Infant Gastrointestinal Digestion: Implications for Oral Antibody Supplementation

Oral administration of engineered immunoglobulins has the potential to prevent enteric pathogen-induced diarrhea in infants. To prevent infection, these antibodies need to survive functionally intact in the proteolytic environment of the gastrointestinal tract. This research examined both ex vivo and in vivo the functional survival across infant digestion of palivizumab, a model FDA-approved recombinant antibody against respiratory syncytial virus (RSV) F protein. Palivizumab-fortified feed (formula or human milk), infant gastric, and intestinal samples were incubated to simulate in vivo digestion (ex vivo digestion). Palivizumab-fortified human milk was also fed to infants, followed by collection of gastric and intestinal samples (in vivo digestion). Palivizumab was purified from the samples of digestate using protein G spin columns followed by filtration through molecular weight cut-off membranes (30 kDa).
Palivizumab functional survival across ex vivo and in vivo digestion was determined via an anti-idiotype ELISA and an RSV plaque reduction neutralization test. Palivizumab concentration and RSV neutralization capacity both decreased when incubated in intestinal samples (ex vivo study). The concentration and neutralization activity of orally-supplemented palivizumab also decreased across infant digestion (in vivo study). These results indicate that if recombinant IgGs were selected for oral supplementation to prevent enteric infections, appropriate dosing would need to account for degradation occurring in the digestive system. Other antibody formats, structural changes, or encapsulation could enhance survival in the infant gastrointestinal tract.

Mapping of Recognition Sites of Monoclonal Antibodies Responsible for the Inhibition of Pneumolysin Functional Activity

The pathogenicity of many bacteria, including Streptococcus pneumoniae, depends on pore-forming toxins (PFTs) that cause host cell lysis by forming large pores in cholesterol-containing cell membranes. Therefore, PFTs-neutralising antibodies may provide useful tools for reducing S. pneumoniae pathogenic effects. This study aimed at the development and characterisation of monoclonal antibodies (MAbs) with neutralising activity to S. pneumoniae PFT pneumolysin (PLY). Five out of 10 produced MAbs were able to neutralise the cytolytic activity of PLY on a lung epithelial cell line. Epitope mapping with a series of recombinant overlapping PLY fragments revealed that neutralising MAbs are directed against PLY loops L1 and L3 within domain 4. The epitopes of MAbs 3A9, 6E5 and 12F11 located at L1 loop (aa 454-471) were crucial for PLY binding to the immobilised cholesterol.
In contrast, the MAb 12D10 recognising L3 (aa 403-423) and the MAb 3F3 against the conformational epitope did not interfere with PLY-cholesterol interaction. Due to conformation-dependent binding, the approach to use overlapping peptides for fine epitope mapping of the neutralising MAbs was unsuccessful. Therefore, the epitopes recognised by the MAbs were analysed using computational methods. This study provides new data on PLY sites involved in functional activity.

Goat Anti-Human Activin RIIB Polyclonal Antibody [Functional Grade]

DPAB-L21507 Creative Diagnostics 25 µg 551.25 EUR

MTR4 (Superkiller Viralicidic Activity 2-like 2 (S. cerevisiae), Dob1, Mtr4, Functional Spliceosome-Associated Protein 118, FSAP118, KIAA0052) (AP)

MBS6427640-01mL MyBiosource 0.1mL 950 EUR

MTR4 (Superkiller Viralicidic Activity 2-like 2 (S. cerevisiae), Dob1, Mtr4, Functional Spliceosome-Associated Protein 118, FSAP118, KIAA0052) (AP)

MBS6427640-5x01mL MyBiosource 5x0.1mL 4120 EUR

MTR4 (Superkiller Viralicidic Activity 2-like 2 (S. cerevisiae), Dob1, Mtr4, Functional Spliceosome-Associated Protein 118, FSAP118, KIAA0052) (APC)

MBS6427641-01mL MyBiosource 0.1mL 950 EUR

MTR4 (Superkiller Viralicidic Activity 2-like 2 (S. cerevisiae), Dob1, Mtr4, Functional Spliceosome-Associated Protein 118, FSAP118, KIAA0052) (APC)

MBS6427641-5x01mL MyBiosource 5x0.1mL 4120 EUR

MTR4 (Superkiller Viralicidic Activity 2-like 2 (S. cerevisiae), Dob1, Mtr4, Functional Spliceosome-Associated Protein 118, FSAP118, KIAA0052) (FITC)

MBS6427643-01mL MyBiosource 0.1mL 950 EUR

MTR4 (Superkiller Viralicidic Activity 2-like 2 (S. cerevisiae), Dob1, Mtr4, Functional Spliceosome-Associated Protein 118, FSAP118, KIAA0052) (FITC)

MBS6427643-5x01mL MyBiosource 5x0.1mL 4120 EUR

MTR4 (Superkiller Viralicidic Activity 2-like 2 (S. cerevisiae), Dob1, Mtr4, Functional Spliceosome-Associated Protein 118, FSAP118, KIAA0052) (HRP)

MBS6427644-01mL MyBiosource 0.1mL 950 EUR

MTR4 (Superkiller Viralicidic Activity 2-like 2 (S. cerevisiae), Dob1, Mtr4, Functional Spliceosome-Associated Protein 118, FSAP118, KIAA0052) (HRP)

MBS6427644-5x01mL MyBiosource 5x0.1mL 4120 EUR

MTR4 (Superkiller Viralicidic Activity 2-like 2 (S. cerevisiae), Dob1, Mtr4, Functional Spliceosome-Associated Protein 118, FSAP118, KIAA0052) (PE)

MBS6427650-01mL MyBiosource 0.1mL 950 EUR

MTR4 (Superkiller Viralicidic Activity 2-like 2 (S. cerevisiae), Dob1, Mtr4, Functional Spliceosome-Associated Protein 118, FSAP118, KIAA0052) (PE)

MBS6427650-5x01mL MyBiosource 5x0.1mL 4120 EUR

Active porcine tPA functional assay

MBS480486-1Kit MyBiosource 1Kit 825 EUR

Active porcine tPA functional assay

MBS480486-5x1Kit MyBiosource 5x1Kit 3755 EUR

MTR4 (Superkiller Viralicidic Activity 2-like 2 (S. cerevisiae), Dob1, Mtr4, Functional Spliceosome-Associated Protein 118, FSAP118, KIAA0052) (Biotin)

MBS6427642-01mL MyBiosource 0.1mL 950 EUR

MTR4 (Superkiller Viralicidic Activity 2-like 2 (S. cerevisiae), Dob1, Mtr4, Functional Spliceosome-Associated Protein 118, FSAP118, KIAA0052) (Biotin)

MBS6427642-5x01mL MyBiosource 5x0.1mL 4120 EUR

Active human uPA functional assay kit

MBS480469-1Kit MyBiosource 1Kit 815 EUR

Active human uPA functional assay kit

MBS480469-5x1Kit MyBiosource 5x1Kit 3700 EUR

Active mouse uPA functional assay kit

MBS480470-1Kit MyBiosource 1Kit 825 EUR

Active mouse uPA functional assay kit

MBS480470-5x1Kit MyBiosource 5x1Kit 3755 EUR

MTR4 (Superkiller Viralicidic Activity 2-like 2 (S. cerevisiae), Dob1, Mtr4, Functional Spliceosome-Associated Protein 118, FSAP118, KIAA0052) (MaxLight 405)

MBS6427645-01mL MyBiosource 0.1mL 950 EUR

MTR4 (Superkiller Viralicidic Activity 2-like 2 (S. cerevisiae), Dob1, Mtr4, Functional Spliceosome-Associated Protein 118, FSAP118, KIAA0052) (MaxLight 405)

MBS6427645-5x01mL MyBiosource 5x0.1mL 4120 EUR

MTR4 (Superkiller Viralicidic Activity 2-like 2 (S. cerevisiae), Dob1, Mtr4, Functional Spliceosome-Associated Protein 118, FSAP118, KIAA0052) (MaxLight 490)

MBS6427646-01mL MyBiosource 0.1mL 950 EUR

MTR4 (Superkiller Viralicidic Activity 2-like 2 (S. cerevisiae), Dob1, Mtr4, Functional Spliceosome-Associated Protein 118, FSAP118, KIAA0052) (MaxLight 490)

MBS6427646-5x01mL MyBiosource 5x0.1mL 4120 EUR

MTR4 (Superkiller Viralicidic Activity 2-like 2 (S. cerevisiae), Dob1, Mtr4, Functional Spliceosome-Associated Protein 118, FSAP118, KIAA0052) (MaxLight 550)

MBS6427647-01mL MyBiosource 0.1mL 950 EUR

MTR4 (Superkiller Viralicidic Activity 2-like 2 (S. cerevisiae), Dob1, Mtr4, Functional Spliceosome-Associated Protein 118, FSAP118, KIAA0052) (MaxLight 550)

MBS6427647-5x01mL MyBiosource 5x0.1mL 4120 EUR

MTR4 (Superkiller Viralicidic Activity 2-like 2 (S. cerevisiae), Dob1, Mtr4, Functional Spliceosome-Associated Protein 118, FSAP118, KIAA0052) (MaxLight 650)

MBS6427648-01mL MyBiosource 0.1mL 950 EUR

Design, creation and in vitro testing of a reduced immunogenicity humanized anti-CD25 monoclonal antibody that retains functional activity

Humanized and fully human sequence-derived therapeutic antibodies retain the capacity to induce anti-drug antibodies. Daclizumab (humanized version of the murine anti-Tac antibody; E.HAT) was selected for a proof of concept application of engineering approaches to reduce potential immunogenicity due to its demonstrated immunogenicity in the clinic. Reduced immunogenicity variants of E.HAT were created by identifying and modifying a CD4+ T cell epitope region in the VH region.
Variant epitope region peptides were selected for their reduced capacity to induce CD4+ T cell proliferative responses in vitro. Variant antibody molecules were created, and CD25 affinity and potency were similar to the unmodified parent antibody. Fab fragments from the variant antibodies induced a lower frequency and magnitude of responses in human peripheral blood mononuclear cells proliferation tests. By the empirical selection of two amino acid mutations, fully functional humanized E.HAT antibodies with reduced potential to induce immune responses in vitro were created.

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