Molecular characteristics of the immune escape of coronavirus PEDV under the pressure of vaccine immunity

ABSTRACT

Coronaviruses have undergone evolutionary changes and mutations in response to the immune pressures exerted by vaccines and environmental factors, resulting in more severe consequences during breakthrough infections. Nevertheless, the specific correlation between the evolutionary mutations of coronaviruses and immune pressures remains ambiguous. Swine coronavirus—porcine epidemic diarrhea virus (PEDV)—has existed for decades. This study utilized in vivo preparation of polyclonal antibodies against the PEDV and identified critical neutralizing epitopes through serial in vitro passaging. Then, the recombinant mutated strains were successfully constructed.

In vitro experiments confirmed the ability of the rA1273P strain to escape neutralization by polyclonal antibodies. Both in vitro cell studies and in vivo animal experiments revealed that the strain maintains virulence and pathogenicity while evading antibody pressure post-vaccination. The pathogenicity of the strain while evading immune pressure is comparable to wild-type strains. A comparison of the S protein gene between vaccine strains and clinical strains identified mutations in 1273 amino acid positions in clinical strains. In conclusion, this study identified a novel PEDV S protein neutralizing site under immune pressure through serial passaging, indicating that the 1,273th amino acid position is prone to mutation under prolonged antibody pressure, enhancing the virus’s ability to escape hosts. This study offers new insights into the interpretation of coronavirus escape immune pressure and provides technical support for monitoring and predicting the variation and evolution of coronavirus.

IMPORTANCE

Coronaviruses represent an ongoing public health threat because of high variability. Since 2010, the emergence of highly pathogenic porcine epidemic diarrhea virus (PEDV) strains has resulted in significant economic losses to the global pig industry. PEDV undergoes evolution and mutation under external immune pressure, rendering it an increasingly challenging target for prevention and control measures. Here, we prepared the polyclonal antibodies against PEDV and identified a novel neutralization epitope on the S protein (1,273th amino acids) through serial in vitro passaging. Furthermore, our findings indicate that the mutation of A1273P in the S protein did not alter the virulence of the PEDV but significantly enhanced its ability to escape and infect the host in vitro and in vivo

. Finally, we found that the 1,273 amino acid position of the S gene has been mutated to varying degrees in clinical PEDV strains. This work provides a specific correlation between the evolutionary mutations of coronaviruses and immune pressures.

Background:

Coronaviruses are known to induce a range of respiratory, gastrointestinal, and central nervous system illnesses in both humans and animals, posing a significant threat to human health and resulting in substantial economic burdens. The coronavirus belongs to the order *Nidovirales*, the family *Coronaviridae*, and the genus *Coronavirus*. Viruses in the genus *Coronavirus* are enveloped and possess a linear single-stranded positive-sense RNA genome. The viral genome primarily encodes four structural proteins: nucleocapsid (N), envelope (E), membrane (M), and spike (S) proteins. The S protein plays a crucial role in facilitating the binding of the virus envelope to host cell receptors through its receptor-binding domain and N-terminal domain, thereby mediating fusion between host cells and the virus. Coronaviruses exhibit a high capacity for adapting to novel environments by means of mutation and recombination, particularly through specific mutations at the S protein binding sites. This capability allows viruses to effectively alter their host range and tissue tropism, while significantly influencing the pathogenicity, transmissibility, and immune responses following host infection. Porcine epidemic diarrhea virus (PEDV), belonging to the genus *Alphacoronavirus*, was first reported in the 1970s. Before 2010, the PEDV G1a classic subgroup in China was limited to sporadic and regional outbreaks, attributed to the extensive use of PEDV CV777 inactivated and attenuated vaccines. However, in October 2010, a highly pathogenic PEDV G2a variant strain emerged in southern Chinese provinces, rapidly spreading nationwide. Even pig farms immunized with the classical strain vaccine experienced a 100% mortality rate in newborn piglets.

Methods:

This study utilized in vivo preparation of polyclonal antibodies against the PEDV and identified critical neutralizing epitopes through serial in vitro passaging. Then, the recombinant mutated strains were successfully constructed. In vitro experiments confirmed the ability of the rA1273P strain to escape neutralization by polyclonal antibodies. Both in vitro cell studies and in vivo animal experiments revealed that the strain maintains virulence and pathogenicity while evading antibody pressure post-vaccination.

Results:

In vitro experiments confirmed the ability of the rA1273P strain to escape neutralization by polyclonal antibodies. Both in vitro cell studies and in vivo animal experiments revealed that the strain maintains virulence and pathogenicity while evading antibody pressure post-vaccination. The pathogenicity of the strain while evading immune pressure is comparable to wild-type strains. A comparison of the S protein gene between vaccine strains and clinical strains identified mutations in 1273 amino acid positions in clinical strains. This study identified a novel PEDV S protein neutralizing site under immune pressure through serial passaging, indicating that the 1,273th amino acid position is prone to mutation under prolonged antibody pressure, enhancing the virus’s ability to escape hosts.

Data Summary:

The 1,273th amino acid position of the S gene has been mutated to varying degrees in clinical PEDV strains. The mutation of A1273P in the S protein did not alter the virulence of the PEDV but significantly enhanced its ability to escape and infect the host in vitro and in vivo.

Conclusions:

This study identified a novel PEDV S protein neutralizing site under immune pressure through serial passaging, indicating that the 1,273th amino acid position is prone to mutation under prolonged antibody pressure, enhancing the virus’s ability to escape hosts. This work provides a specific correlation between the evolutionary mutations of coronaviruses and immune pressures.

Practical Significance:

Coronaviruses represent an ongoing public health threat because of high variability. Since 2010, the emergence of highly pathogenic porcine epidemic diarrhea virus (PEDV) strains has resulted in significant economic losses to the global pig industry. This study offers new insights into the interpretation of coronavirus escape immune pressure and provides technical support for monitoring and predicting the variation and evolution of coronavirus.