CRRSP61 Antibody

What is CR6261 and what is its significance in influenza research?

CR6261 is a broadly neutralizing human antibody that was isolated from the immune repertoire of a healthy, vaccinated individual using phage display selection on recombinant H5 HA, despite no known prior exposure to H5 viruses . The antibody's significance lies in its extraordinary capacity to neutralize multiple influenza subtypes, including H1, H2, H5, H6, H8, and H9, and to protect mice from lethal challenge with H1N1 and H5N1 viruses when administered up to five days post-infection .

Unlike conventional influenza antibodies that target the hypervariable regions surrounding the receptor binding site on the HA head, CR6261 binds to a highly conserved helical region in the membrane-proximal stem of HA1/HA2 . This unique binding characteristic enables it to interfere with membrane fusion rather than receptor binding, representing an alternative mechanism of neutralization . The conservation of this epitope across various influenza subtypes suggests CR6261 could inform the development of universal influenza vaccines or antibody-based therapies with broad-spectrum protection against current and potentially future pandemic influenza strains .

How is CR6261's epitope structurally characterized?

CR6261 recognizes a conformational epitope located at the membrane-proximal end of the HA molecule, approximately 25 Å from the viral membrane . Crystal structures determined at 2.2 Å and 2.7 Å resolutions for CR6261 Fab in complexes with HA from the 1918 H1N1 pandemic virus and a lethal H5N1 avian influenza virus have revealed the precise nature of this interaction .

The epitope comprises two component parts: (i) the HA2 A-helix, which accounts for most of the interacting surface and polar contacts, and (ii) the adjacent HA1 region, which makes primarily hydrophobic contacts with CR6261 . Interestingly, the interaction is mediated exclusively by the antibody's heavy chain, with no contribution from the light chain - an unusual feature in antibody-antigen recognition . The binding interface is dominated by the heavy-chain complementarity-determining regions HCDR1 and HCDR2, with HCDR3 making relatively minor contributions .

This structural characterization has revealed that residues in the A-helix that mediate hydrogen bonds with CR6261 also form hydrogen bonds and a salt bridge to the HA2 C-terminal H segment in the post-fusion conformation, suggesting the epitope plays a critical role in the membrane fusion process .

What genetic factors contribute to CR6261's broad neutralization capacity?

The broad neutralization capacity of CR6261 is closely tied to its genetic origins. The antibody is derived from the V​H1-69 germline segment, which codes for a conserved hydrophobic tip on HCDR2 . This genetic association appears significant, as 12 out of 13 broadly neutralizing clones isolated alongside CR6261 were also derived from V​H1-69 .

The HCDR2 region of CR6261 inserts into a hydrophobic pocket adjacent to the A-helix in both H1 and H5 HA structures, providing critical binding energy . Additionally, HCDR1, which makes the most important contacts with the highly conserved A-helix, is also germline-encoded . While somatic mutations in the V​H1-69 framework make important contributions to binding affinity, the HCDR3 region contributes relatively little to antigen recognition - a notable deviation from the usual dominant role of HCDR3 in most antibodies .

This genetic profile suggests that the human immune repertoire possesses inherent potential to generate broadly neutralizing influenza antibodies through the V​H1-69 germline segment, though the frequency of such antibodies in natural immune responses appears limited . Understanding these genetic constraints may inform strategies to elicit similar broadly neutralizing antibodies through vaccination.

How does CR6261 inhibit the membrane fusion process?

CR6261 employs a distinctive mechanism of viral neutralization compared to conventional anti-influenza antibodies. While most neutralizing antibodies target the hypervariable regions surrounding the receptor binding site and interfere with host cell attachment, CR6261 does not inhibit agglutination of erythrocytes, indicating it doesn't block receptor binding . Instead, it neutralizes the virus by preventing the conformational changes in HA required for membrane fusion .

The crystal structures of CR6261-HA complexes reveal that the antibody binds at the membrane-proximal stem of HA, approximately parallel to the viral envelope plane . At this position, CR6261 acts as a molecular buttress that opposes the dissociation of the HA1 heads from HA2 during the fusion process . Specifically, structural analysis identified a pivot region where HA1 would typically peel away from HA2 during fusion, with CR6261 strategically positioned below this hinge .

This mechanism explains why CR6261 Fab fragments neutralize virus as potently as intact IgG - because the inhibition relies on direct blocking of conformational changes rather than cross-linking adjacent HA protomers . The epitope's conservation across multiple influenza subtypes suggests this region plays a critical functional role in membrane fusion that cannot easily mutate without compromising viral fitness, making it an attractive target for broad-spectrum antiviral strategies .

What are the potential autoreactivity concerns with CR6261?

A significant consideration for the therapeutic application of CR6261 is its potential for autoreactivity with human tissues. Comprehensive screening using normal human tissue microarrays comprising 30 different tissues revealed that CR6261 shows reactivity with human tissues, particularly with pituitary gland tissue . This finding raises important safety considerations for its potential use as a therapeutic agent.

More specifically, protein array analysis identified high-affinity interaction between CR6261 and the autoantigen "Enhancer of mRNA decapping 3 homolog" (EDC3), an interaction not previously described in earlier characterizations of this antibody . The table below summarizes the tissue reactivity profile of CR6261 compared to other broadly neutralizing antibodies:

*Values represent mean reactivity scores (n=3) with higher numbers indicating stronger reactivity

Competition experiments demonstrated that EDC3 binding to CR6261 blocked the antibody's ability to bind to influenza hemagglutinin in surface plasmon resonance (SPR) assays . This competitive binding suggests molecular mimicry between EDC3 and the viral epitope, which has important implications for both therapeutic development and understanding potential adverse effects . These findings underscore the need for careful evaluation of broadly neutralizing antibodies like CR6261 for therapeutic applications and vaccine strategies aimed at generating similar antibodies.

How can CR6261's epitope inform universal influenza vaccine design?

The identification of CR6261's conserved epitope represents a significant advancement toward the development of a universal influenza vaccine. The high conservation of the stem region targeted by CR6261 across multiple influenza subtypes suggests that antigens designed to elicit similar antibodies could provide broad protection against diverse influenza viruses, including potential pandemic strains .

Although CR6261 recognizes a conformational epitope between HA1 and HA2, vaccine design efforts may focus primarily on mimicking the A-helix component, which accounts for most of the interacting surface and polar contacts . This might be achievable through the design of linear peptide antigens, though novel strategies would likely be required to improve immunogenicity, as this epitope is not particularly exposed in intact virus particles .

Several research approaches could be considered for vaccine design based on CR6261's epitope:

• Structure-based design of immunogens that present the conserved stem epitope in an optimal conformation

• Prime-boost strategies that preferentially expand B cells targeting the conserved stem region

• Germline-targeting approaches that specifically activate V​H1-69-expressing B cells

• Chimeric hemagglutinin constructs that focus the immune response on the stem by presenting novel head domains

The challenge remains that this epitope is not immunodominant under natural infection or conventional vaccination . Additionally, the potential autoreactivity of CR6261-like antibodies adds another layer of complexity that must be addressed in vaccine development . Nevertheless, the CR6261 epitope marks an important step toward the development of a durable and cross-protective "universal" vaccine against influenza A .

What methodologies are optimal for assessing CR6261 neutralization activity?

Accurate assessment of CR6261's neutralization activity requires robust and reliable methodologies. Based on the available research, several approaches have proven effective for evaluating the neutralization potency of CR6261 and similar broadly neutralizing antibodies.

In one study examining various neutralizing antibodies, researchers found a strong positive correlation (Pearson r = 0.94, p < 0.001) between IC80 values determined by ELISA and fluorescent reporter-based readouts . Similarly, when comparing reporter viruses (R3ΔPB1) with parental viruses in neutralization assays, the IC80 values showed positive correlation (Pearson r = 0.87, p < 0.001), indicating that reporter systems can reliably measure neutralization activity while offering increased safety and throughput .

For assessing EDC3 competition with hemagglutinin binding, surface plasmon resonance (SPR) has proven valuable . Specifically, competition experiments can be performed by injecting proteins (at 20 μg/mL) for 180 seconds at 50 μL/min on antibody-coated sensor chips, followed by injection of recombinant HA (10 μg/mL) for association . The percentage of HA binding remaining in the presence of competing proteins can then be calculated to quantify the degree of competition .

These methodologies provide researchers with reliable tools to characterize the neutralization properties of CR6261 and similar antibodies, facilitating the development of therapeutic strategies and vaccines based on their epitopes.