CoV Spike Bovine

What is the homology between bovine coronavirus spike protein and SARS-CoV-2 spike protein?

The amino acid sequence homology between bovine coronavirus spike protein (accession no. AAA66399.1) and SARS-CoV-2 spike proteins is surprisingly low, with only 29.59% homology to the Wuhan strain and 29.27% to the Omicron variant . Despite this relatively low sequence homology, there appears to be functional and immunological cross-reactivity between these proteins, suggesting that three-dimensional structural similarities or conserved epitopes may be more relevant than linear sequence identity.

How do SARS-CoV-2 variants differ in their ability to use bovine ACE2 as a receptor?

Table 1: Relative Binding Affinities of SARS-CoV-2 RBDs to ACE2 Proteins from Different Species

The binding affinities of cattle ACE2 proteins to the RBDs of these VOCs became comparable to that of human ACE2 to the early WHU01 strain, suggesting that cattle might have become more susceptible to infection with newer variants .

What evidence exists for cross-reactive immunity between bovine coronavirus and SARS-CoV-2?

Substantial evidence suggests bovine coronavirus spike protein (BoS) may represent a source of protective immunity to SARS-CoV-2. In experimental studies, vaccination of BALB/c mice with a Bovine herpesvirus 4 (BoHV-4)-based vector expressing BoS induced both cell-mediated and humoral immune responses that cross-react with SARS-CoV-2 spike protein . While the spike-specific antibodies induced by BoS did not neutralize SARS-CoV-2, the T lymphocytes activated by BoS were able to induce cytotoxicity of cells expressing spike proteins derived from several SARS-CoV-2 variants . This demonstrates that cross-reactive T-cell immunity may exist even in the absence of neutralizing antibodies.

How can researchers test cross-reactivity between bovine coronavirus antibodies and SARS-CoV-2 spike variants?

To test cross-reactivity between bovine coronavirus antibodies and SARS-CoV-2 spike variants, researchers have employed direct enzyme-linked immunosorbent assays (ELISAs). This approach involves testing whether bovine whey IgG enriched fractions contain antibodies against recombinant partial length spike proteins of different SARS-CoV-2 variants .

A methodical approach involves:

• Express and purify recombinant partial length spike proteins representing various regions (e.g., aa 177–512, aa 509–685, aa 177–324, aa 250–410, and aa 387–516) from different SARS-CoV-2 variants

• Coat ELISA plates with these protein fragments

• Incubate with bovine IgG samples

• Detect bound antibodies using species-specific secondary antibodies

• Compare binding profiles across different spike regions and variants

This methodology allows for assessment of cross-reactivity against specific regions of the spike protein from different SARS-CoV-2 variants, enabling the identification of conserved epitopes .

What animal models are suitable for studying bovine coronavirus spike-induced immunity against SARS-CoV-2?

BALB/c mice have proven to be an effective model for studying whether vaccination with bovine coronavirus spike proteins induces cross-reactive immunity against SARS-CoV-2 . This model is particularly valuable because it allows researchers to assess both antibody responses and T-cell responses. In studies using this model, researchers have demonstrated that while antibodies induced by bovine coronavirus spike weren't neutralizing against SARS-CoV-2, T-cells showed cross-reactive cytotoxicity .

When designing animal models for such studies, researchers should consider:

• Expressing bovine coronavirus spike protein using appropriate vectors (e.g., BoHV-4-based vectors)

• Assessing humoral immunity through antibody binding and neutralization assays

• Evaluating cellular immunity through T-cell activation and cytotoxicity assays

• Testing cross-reactivity against multiple SARS-CoV-2 variants

• Performing challenge studies when appropriate to assess protective efficacy

This comprehensive approach allows for a thorough assessment of both arms of the adaptive immune response following bovine coronavirus spike immunization.

How can researchers evaluate the inhibitory potential of bovine milk-derived components against SARS-CoV-2?

Evaluating the inhibitory potential of bovine milk-derived components against SARS-CoV-2 requires a multi-faceted methodological approach. Research has shown that bovine lactoferrin (bLf) inhibits SARS-CoV-2 through dual mechanisms: blocking the spike protein-ACE2 interaction and interfering with viral RNA-dependent RNA polymerase (RdRp) activity .

A comprehensive evaluation protocol would include:

• Protein-protein interaction assays to assess binding between milk components and SARS-CoV-2 spike RBD

• Cell-based entry assays using pseudotyped viruses expressing SARS-CoV-2 spike proteins

• Enzymatic assays to evaluate inhibition of viral RdRp activity

• Live virus neutralization assays under appropriate biosafety conditions

• In vivo studies to assess therapeutic efficacy, viral load reduction, and improvement in pathological changes

This methodological approach enables researchers to identify and characterize milk-derived components with potential antiviral properties against SARS-CoV-2 and understand their mechanisms of action.

How do we interpret differences in antibody versus T-cell responses in cross-reactive immunity studies?

The interpretation of differential antibody versus T-cell responses in cross-reactive immunity studies requires careful consideration of the distinct nature of these immune responses. Research with bovine coronavirus spike protein demonstrates an interesting case where immunization induced antibodies that could bind to SARS-CoV-2 spike but couldn't neutralize the virus, while simultaneously generating T-cells capable of recognizing and killing cells expressing SARS-CoV-2 spike variants .

This differential response can be interpreted through several lenses:

• Epitope specificity: Antibodies may recognize non-neutralizing epitopes shared between bovine coronavirus and SARS-CoV-2 spikes, while T-cells may recognize conserved peptides that, when presented on MHC, trigger effective responses.

• Functional outcomes: Binding antibodies may contribute to immunity through mechanisms other than neutralization (e.g., antibody-dependent cellular cytotoxicity or complement activation).

• Protective potential: T-cell responses might provide significant protection even in the absence of neutralizing antibodies, particularly against severe disease.

• Vaccine design implications: Vaccines designed to elicit both humoral and cellular immunity may provide broader protection against coronavirus variants.

This underscores the importance of comprehensively assessing both arms of adaptive immunity when evaluating cross-reactive responses between coronaviruses.

What explains the presence of SARS-CoV-2 cross-reactive antibodies in pre-pandemic bovine milk samples?

The intriguing presence of SARS-CoV-2 cross-reactive antibodies in pre-pandemic bovine milk samples (collected in 2018 and 2019) requires careful interpretation. Bovine whey IgG enriched fractions from these samples contained antibodies that recognized SARS-CoV-2 spike proteins, with highest reactivity against the aa 177–512 region of the Omicron spike protein .

Several explanations for this phenomenon are possible:

This finding suggests that natural exposure to animal coronaviruses may generate antibodies with unexpected cross-reactivity to novel human coronaviruses.

How significant are RBD mutations in changing the host tropism of SARS-CoV-2 variants?

RBD mutations play a critical role in altering the host tropism of SARS-CoV-2 variants. Research demonstrates that mutations in SARS-CoV-2 variants, particularly those found in VOCs, significantly enhanced binding to ACE2 from species previously thought to be less susceptible, including cattle and pigs .

The significance of these mutations manifests in several ways:

• Expanded host range: VOCs with mutations like N501Y demonstrate significantly enhanced affinities to cattle, pig, and mouse ACE2 proteins compared to early isolates .

• Structural adaptations: Spike trimers of multiple SARS-CoV-2 variants, including VOCs, have a higher propensity to adopt an "RBD-up" or open state than earlier variants, further enabling efficient usage of non-human ACE2 receptors .

• Evolutionary trajectory: These changes suggest SARS-CoV-2 was evolving toward better utilization of ACE2 across species, through both increasing RBD affinity and improving RBD accessibility .

• Surveillance implications: Enhanced ability to use bovine ACE2 suggests that cattle might have become more susceptible to newer SARS-CoV-2 variants, necessitating closer monitoring of these livestock species .

These findings highlight the dynamic nature of SARS-CoV-2 host tropism and the importance of monitoring RBD mutations for assessing potential cross-species transmission risks.

What mechanisms explain the higher affinity of VOC RBDs for bovine ACE2 compared to early SARS-CoV-2 strains?

The enhanced affinity of Variant of Concern (VOC) RBDs for bovine ACE2 compared to early SARS-CoV-2 strains involves multiple molecular mechanisms. VOCs like B.1.1.7 (Alpha) carry mutations such as N501Y in the RBD region that significantly alter interaction with ACE2 receptors across species .

Several mechanisms likely contribute to this phenomenon:

• Direct binding interface modifications: Mutations like N501Y occur at the RBD-ACE2 interface and may create more favorable interactions with bovine ACE2 residues compared to the original tyrosine.

• Conformational changes: VOC mutations may alter the RBD conformation in ways that better accommodate the structural differences between human and bovine ACE2.

• Enhanced RBD accessibility: Structural studies have shown that spike trimers of multiple SARS-CoV-2 variants have a significantly higher propensity to adopt an "RBD-up" or open state than earlier variants, making the ACE2-binding surface more accessible .

• Allosteric effects: Mutations outside the direct binding interface may cause allosteric changes that indirectly enhance RBD-ACE2 interactions.

The combination of these factors—specific amino acid changes enhancing direct ACE2 interaction and conformational changes making RBDs more accessible—likely explains the increased affinity of VOCs for bovine ACE2.

How might bovine lactoferrin inhibit SARS-CoV-2 through dual mechanisms?

Bovine lactoferrin (bLf) exhibits a fascinating dual mechanism of action against SARS-CoV-2, targeting both viral entry and replication. Research demonstrates that bLf inhibits SARS-CoV-2 by combining with the spike protein RBD and inhibiting the viral RNA-dependent RNA polymerase (RdRp) activity .

The dual inhibitory mechanisms can be explained as follows:

• Entry inhibition: bLf likely binds to the SARS-CoV-2 spike protein RBD, preventing its interaction with the ACE2 receptor on host cells. This mechanism blocks the initial attachment and entry step of the viral infection cycle.

• Replication inhibition: In a novel finding, bLf interferes with SARS-CoV-2 RdRp activity . As RdRp is essential for viral genome replication and transcription, this inhibition prevents viral multiplication within infected cells.

• Conserved target advantage: RdRp is highly conserved among coronaviruses, making it an excellent target for broad-spectrum antivirals. This explains why bLf can inhibit both SARS-CoV-2 and other coronaviruses.

• Immune modulation: Beyond direct antiviral effects, bLf can induce immune mediators that interrupt viral infections, providing an additional layer of protection .

This multi-target approach may explain why bovine lactoferrin shows promise as a potential therapeutic against SARS-CoV-2, addressing both viral entry and replication processes.

What are the potential applications of bovine coronavirus spike protein in SARS-CoV-2 vaccine development?

The cross-reactive immune responses induced by bovine coronavirus spike protein (BoS) present intriguing opportunities for SARS-CoV-2 vaccine development. Research demonstrates that BoS vaccination induces both humoral and cellular immune responses cross-reactive with SARS-CoV-2 .

Potential applications in vaccine development include:

• Cross-protective immunity: Using BoS as an immunogen could potentially induce T-cell responses that recognize conserved epitopes across coronavirus species, providing broader protection against emerging variants and potentially future coronavirus outbreaks .

• T-cell focused vaccines: The finding that BoS induces cross-reactive T-cell responses capable of killing cells expressing SARS-CoV-2 spike proteins, even without neutralizing antibodies, suggests potential for T-cell-focused vaccine strategies .

• Chimeric spike constructs: Creating chimeric proteins that combine the most cross-reactive regions from bovine coronavirus spike with SARS-CoV-2-specific regions could potentially elicit broader immunity.

• Pre-existing immunity leverage: Understanding how exposure to bovine coronaviruses might contribute to pre-existing cross-reactive immunity could inform population-level vaccination strategies.

• Veterinary applications: Given the increasing susceptibility of cattle to newer SARS-CoV-2 variants, BoS-based vaccines might protect livestock from potential SARS-CoV-2 infections .

These applications highlight how comparative coronavirus research could contribute to novel vaccine approaches with broader spectrum protection.

How do we reconcile the low sequence homology between bovine coronavirus and SARS-CoV-2 spike proteins with observed immunological cross-reactivity?

The apparent contradiction between low sequence homology (~29.5%) between bovine coronavirus and SARS-CoV-2 spike proteins and their demonstrated immunological cross-reactivity presents a fascinating scientific puzzle . Several mechanisms might explain this phenomenon:

This apparent contradiction highlights the complexity of immunological cross-reactivity and the limitations of predicting cross-protection based solely on sequence homology.

What explains the contradictory findings regarding neutralizing versus binding antibodies in cross-reactivity studies?

The discrepancy between binding and neutralizing capabilities of cross-reactive antibodies represents an important immunological nuance in coronavirus research. Studies show that bovine coronavirus spike immunization induced antibodies that could bind to SARS-CoV-2 spike but couldn't neutralize the virus . Similarly, pre-pandemic bovine whey contained antibodies that recognized SARS-CoV-2 spike proteins without demonstrating neutralizing activity .

Several factors may explain this contradiction:

• Epitope location and function: Antibodies may recognize regions of the spike protein not directly involved in receptor binding or fusion, allowing binding without interfering with viral entry mechanisms.

• Binding affinity threshold: Neutralization typically requires higher antibody-antigen binding affinity than simple detection in an ELISA. Cross-reactive antibodies may bind with sufficient affinity for detection but insufficient strength for neutralization.

• Conformational requirements: Effective neutralizing antibodies often recognize the spike protein in specific conformations required for function. Cross-reactive antibodies may recognize shared epitopes in detection assays but not in the conformation required for neutralization.

• Antibody concentration: Higher concentrations of lower-affinity antibodies might be required for neutralization than were present in the studies.

• Accessibility in native conformation: Some epitopes recognized in assays using recombinant proteins may be inaccessible in the native viral spike trimer conformation.

This distinction is critical for vaccine development and understanding protective immunity, as binding antibodies may still contribute to protection through mechanisms like antibody-dependent cellular cytotoxicity or complement activation.

What implications do species-specific ACE2 affinity changes have for zoonotic transmission risk assessment?

The evolving ability of SARS-CoV-2 variants to utilize ACE2 from different species has profound implications for zoonotic transmission risk assessment. Research demonstrates that VOCs show enhanced binding to cattle and pig ACE2 compared to early strains , suggesting a potentially expanded host range.

These findings carry several important implications:

• Expanded reservoir potential: Enhanced ability to use bovine and porcine ACE2 suggests these livestock species might become susceptible to infection with newer variants, potentially establishing new animal reservoirs .

• Bidirectional zoonotic risk: If cattle become infected with SARS-CoV-2 variants, there's potential for viral adaptation and possible transmission back to humans, potentially with new mutations.

• Surveillance priorities: These findings suggest that livestock species previously considered at low risk should now be included in SARS-CoV-2 surveillance programs, particularly in regions with high prevalence of VOCs .

• Agricultural biosecurity: Enhanced susceptibility of livestock necessitates reevaluation of biosecurity measures to prevent human-to-animal transmission in agricultural settings.

• Evolutionary pressure: Circulation in new hosts could subject SARS-CoV-2 to different evolutionary pressures, potentially leading to new variants with altered transmissibility, pathogenicity, or immunogenicity.

This evolving host tropism underscores the dynamic nature of zoonotic transmission risk and highlights the need for continued monitoring of SARS-CoV-2 variants in multiple animal species.