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Background: Evolutionary pressure has led to the emergence of SARS-CoV-2 variants, with the most recent Omicron variant containing an unparalleled 30 mutations in the spike protein. Many of these mutations are expected to increase immune evasion, thus making breakthrough cases and re-infection more common., Methods: From June 2020 to December 2021 serial blood samples (initial post recovery, 6 months, 12 months) were collected from a COVID-19 convalescent cohort in Boston, MA. Plasma was isolated for use in Mesoscale Discovery based antibody binding assays. Unvaccinated donors or those vaccinated prior to the primary blood draw were excluded from this analysis, as were those who did not have at least two blood draws. Wilcoxon signed rank tests were used to compare pre- and post-vaccination titers and antibody response against different variants, while McNemar tests were used to compare the proportions of achieving ≥ 4 fold increases against different variants., Findings: Forty-eight COVID convalescent donors with post-infection vaccination (hybrid immunity) were studied to evaluate the levels of cross-reactive antibodies pre- and post- vaccination against various SARS-CoV-2 Spike and receptor binding domain (RBD) proteins. Vaccination with BNT162b2, mRNA-1273 or Ad26.COV2.S led to a 6·3 to 7·8 fold increase in anti-Spike antibody titers and a 7·0 to 7·4 fold increase in anti-WT, Alpha and Delta RBD antibody. However, a lower response was observed for Beta and Omicron RBDs with only 7/48 (15%) and 15/48 (31%) donors having a ≥4 fold increase in post-vaccination titers against Beta and Omicron RBDs. Structural analysis of the Beta and Omicron RBDs reveal a shared immune escape strategy involving residues K417-E484-N501 that is exploited by these variants of concern., Interpretation: Through mutations of the K417-E484-N501 triad, SARS-CoV-2 has evolved to evade neutralization by the class I/II anti-RBD antibody fraction of hybrid immunity plasma as the polyclonal antibody response post-vaccination shows limitations in the ability to solve the structural requirements to bind the mutant RBDs., Funding: Massachusetts Consortium on Pathogen Readiness (280870.5116709.0016) and the National Institute of Allergy and Infectious Diseases (1R01AI161152-01A1)., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Porcine epidemic diarrhea virus (PEDV) causes high mortality in neonatal piglets; however, effective and safe vaccines are still not available. We hypothesized that inactivation of the 2'- O -methyltransferase (2'- O -MTase) activity of nsp16 and the endocytosis signal of the spike protein attenuates PEDV yet retains its immunogenicity in pigs. We generated a recombinant PEDV, KDKE 4A , with quadruple alanine substitutions in the catalytic tetrad of the 2'- O -MTase using a virulent infectious cDNA clone, icPC22A, as the backbone. Next, we constructed another mutant, KDKE 4A -SYA, by abolishing the endocytosis signal of the spike protein of KDKE 4A Compared with icPC22A, the KDKE 4A and KDKE 4A -SYA mutants replicated less efficiently in vitro but induced stronger type I and type III interferon responses. The pathogenesis and immunogenicities of the mutants were evaluated in gnotobiotic piglets. The virulence of KDKE 4A -SYA and KDKE 4A was significantly reduced compared with that of icPC22A. Mortality rates were 100%, 17%, and 0% in the icPC22A-, KDKE 4A -, and KDKE 4A -SYA-inoculated groups, respectively. At 21 days postinoculation (dpi), all surviving pigs were challenged orally with a high dose of icPC22A. The KDKE 4A -SYA- and KDKE 4A -inoculated pigs were protected from the challenge, because no KDKE 4A -SYA- and one KDKE 4A -inoculated pig developed diarrhea whereas all the pigs in the mock-inoculated group had severe diarrhea, and 33% of them died. Furthermore, we serially passaged the KDKE 4A -SYA mutant in pigs three times and did not find any reversion of the introduced mutations. The data suggest that KDKE 4A -SYA may be a PEDV vaccine candidate. IMPORTANCE PEDV is the most economically important porcine enteric viral pathogen and has caused immense economic losses in the pork industries in many countries. Effective and safe vaccines are desperately required but still not available. 2'- O -MTase (nsp16) is highly conserved among coronaviruses (CoVs), and the inactivation of nsp16 in live attenuated vaccines has been attempted for several betacoronaviruses. We show that inactivation of both 2'- O -MTase and the endocytosis signal of the spike protein is an approach to designing a promising live attenuated vaccine for PEDV. The in vivo passaging data also validated the stability of the KDKE 4A -SYA mutant. KDKE 4A -SYA warrants further evaluation in sows and their piglets and may be used as a platform for further optimization. Our findings further confirmed that nsp16 can be a universal target for CoV vaccine development and will aid in the development of vaccines against other emerging CoVs., (Copyright © 2019 American Society for Microbiology.)

Porcine reproductive and respiratory syndrome virus (PRRSV) has the ability to suppress the type I interferons (IFNs-α/β) induction to facilitate its survival during infection, and the nsp1 protein of PRRSV has been identified as the potent IFN antagonist. The nsp1β subunit of nsp1 has also been shown to block the host mRNA nuclear export as one of the mechanisms to suppress host antiviral protein production. The SAP motif in nsp1β is the functional motif for both IFN suppression and host mRNA nuclear retention, and using infectious clones, two mutant viruses vL126A and vL135A have been generated. These mutants retain the infectivity, but the phenotype is negative for both IFN suppression and host mRNA nuclear retention due to the loss of the SAP motif. To examine the pathogenic role of IFN suppression in pigs, 40 piglets were allotted to four groups and each group was intramuscularly infected with vL126A, vL135A, wild-type (WT) PRRSV, and placebo. Pigs infected with vL126A or vL135A exhibited mild clinical signs with low viral titers and short duration of viremia. The levels of PRRSV-specific antibody remained comparable in all infected groups but the neutralizing antibody titers were high in vL126A-infected or vL135A-infected pigs. The IFN-α concentration was also high in pigs infected with the SAP mutants. Reversion to WT sequence was observed in the SAP motif in some animals, and the revertants regained the function to suppress IFN production and host mRNA nuclear export, indicating strong selection pressure in the SAP motif of nsp1β. Together, our data demonstrate that the IFN antagonism and host mRNA nuclear retention mediated by nsp1β contributes to viral virulence, and loss of these functions confers PRRSV attenuation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Porcine reproductive and respiratory syndrome (PRRS) remains one of the most economically significant diseases in the swine industry worldwide. The current vaccines are less satisfactory to confer protections from heterologous infections and long-term persistence, and the need for better vaccines are urgent. The immunological hallmarks in PRRSV-infected pigs include the unusually poor production of type I interferons (IFNs-α/β) and the aberrant and delayed adaptive immune responses, indicating that PRRSV has the ability to suppress both innate and adaptive immune responses in the host. Type I IFNs are the potent antiviral cytokines and recent studies reveal their pleiotropic functions in the priming of expansion and maturation of adaptive immunity. Thus, IFN antagonism-negative PRRSV is hypothesized to be attenuated and to build effective and broad- spectrum innate and adaptive immune responses in pigs. Such vaccines are promising alternatives to traditional vaccines for PRRSV., (Copyright © 2017 Elsevier B.V. All rights reserved.)

BACKGROUND: Evolutionary pressure has led to the emergence of SARS-CoV-2 variants, with the most recent Omicron variant containing an unparalleled 30 mutations in the spike protein. Many of these mutations are expected to increase immune evasion, thus making breakthrough cases and re-infection more common. METHODS: From June 2020 to December 2021 serial blood samples (initial post recovery, 6 months, 12 months) were collected from a COVID-19 convalescent cohort in Boston, MA. Plasma was isolated for use in Mesoscale Discovery based antibody binding assays. Unvaccinated donors or those vaccinated prior to the primary blood draw were excluded from this analysis, as were those who did not have at least two blood draws. Wilcoxon signed rank tests were used to compare pre- and post-vaccination titers and antibody response against different variants, while McNemar tests were used to compare the proportions of achieving ≥ 4 fold increases against different variants. FINDINGS: Forty-eight COVID convalescent donors with post-infection vaccination (hybrid immunity) were studied to evaluate the levels of cross-reactive antibodies pre- and post- vaccination against various SARS-CoV-2 Spike and receptor binding domain (RBD) proteins. Vaccination with BNT162b2, mRNA-1273 or Ad26.COV2.S led to a 6·3 to 7·8 fold increase in anti-Spike antibody titers and a 7·0 to 7·4 fold increase in anti-WT, Alpha and Delta RBD antibody. However, a lower response was observed for Beta and Omicron RBDs with only 7/48 (15%) and 15/48 (31%) donors having a ≥4 fold increase in post-vaccination titers against Beta and Omicron RBDs. Structural analysis of the Beta and Omicron RBDs reveal a shared immune escape strategy involving residues K417-E484-N501 that is exploited by these variants of concern. INTERPRETATION: Through mutations of the K417-E484-N501 triad, SARS-CoV-2 has evolved to evade neutralization by the class I/II anti-RBD antibody fraction of hybrid immunity plasma as the polyclonal antibody response post-vaccination shows limitations in the ability to solve the structural requirements to bind the mutant RBDs. FUNDING: Massachusetts Consortium on Pathogen Readiness (280870.5116709.0016) and the National Institute of Allergy and Infectious Diseases (1R01AI161152-01A1).