Live-virus neutralization of the omicron variant in children and adults 14 months after SARS-CoV-2 wild-type infection.

Data on cross-neutralization of the SARS-CoV-2 omicron variant more than 1 year after SARS-CoV-2 infection are urgently needed, especially in children, to predict the likelihood of reinfection and to guide vaccination strategies. In a prospective observational cohort study, we evaluated live-virus neutralization of the SARS-CoV-2 omicron (BA.1) variant in children compared with adults 14 months after mild or asymptomatic wild-type SARS-CoV-2 infection. We also evaluated immunity to reinfection conferred by previous infection plus COVID-19 mRNA vaccination. We studied 36 adults and 34 children 14 months after acute SARS-CoV-2 infection. While 94% of unvaccinated adults (16/17) and children (32/34) neutralized the delta (B.1.617.2) variant, only 1/17 (5.9%) unvaccinated adults, 0/16 (0%) adolescents and 5/18 (27.8%) children <12 years of age had neutralizing activity against omicron (BA.1). In convalescent adults, one or two doses of mRNA vaccine increased delta and omicron neutralization 32-fold, similar to a third mRNA vaccination in uninfected adults. Neutralization of omicron was 8-fold lower than that of delta in both groups. In conclusion, our data indicate that humoral immunity induced by previous SARS-CoV-2 wild-type infection more than 1 year ago is insufficient to neutralize the current immune escape omicron variant.

Immune Response to COVID-19 mRNA Vaccination in Previous Nonresponder Kidney Transplant Recipients After Short-term Withdrawal of Mycophenolic Acid 1 and 3 Months After an Additional Vaccine Dose.

BACKGROUND: The impaired immune response to coronavirus disease 2019 (COVID-19) vaccination in kidney transplant recipients (KTRs) leads to an urgent need for adapted immunization strategies. METHODS: Sixty-nine KTRs without seroconversion after ≥3 COVID-19 vaccinations were enrolled, and humoral response was determined after an additional full-dose mRNA-1273 vaccination by measuring severe acute respiratory syndrome coronavirus 2-specific antibodies and neutralizing antibody activity against the Delta and Omicron variants 1 and 3 mo postvaccination. T-cell response was analyzed 3 mo postvaccination by assessing interferon-γ release. Mycophenolic acid (MPA) was withdrawn in 41 KTRs 1 wk before until 4 wk after vaccination to evaluate effects on immunogenicity. Graft function, changes in donor-specific anti-HLA antibodies, and donor-derived cell-free DNA were monitored in KTRs undergoing MPA withdrawal. RESULTS: Humoral response to vaccination was significantly stronger in KTRs undergoing MPA withdrawal 1 mo postvaccination; however, overall waning humoral immunity was noted in all KTRs 3 mo after vaccination. Higher anti-S1 immunoglobulin G levels correlated with better neutralizing antibody activity against the Delta and Omicron variants, whereas no significant association was detected between T-cell response and neutralizing antibody activity. No rejection occurred during study, and graft function remained stable in KTRs undergoing MPA withdrawal. In 22 KTRs with Omicron variant breakthrough infections, neutralizing antibody activity was better against severe acute respiratory syndrome coronavirus 2 wild-type and the Delta variants than against the Omicron variant. CONCLUSIONS: MPA withdrawal to improve vaccine responsiveness should be critically evaluated because withdrawing MPA may be associated with enhanced alloimmune response, and the initial effect of enhanced seroconversion rates in KTRs with MPA withdrawal disappears 3 mo after vaccination.

Humoral response to SARS-CoV-2 mRNA vaccination in previous non-responder kidney transplant recipients after short-term withdrawal of mycophenolic acid.

Seroconversion rates after COVID-19 vaccination are significantly lower in kidney transplant recipients compared to healthy cohorts. Adaptive immunization strategies are needed to protect these patients from COVID-19. In this prospective observational cohort study, we enrolled 76 kidney transplant recipients with no seroresponse after at least three COVID-19 vaccinations to receive an additional mRNA-1273 vaccination (full dose, 100 µg). Mycophenolic acid was withdrawn in 43 selected patients 5-7 days prior to vaccination and remained paused for 4 additional weeks after vaccination. SARS-CoV-2-specific antibodies and neutralization of the delta and omicron variants were determined using a live-virus assay 4 weeks after vaccination. In patients with temporary mycophenolic acid withdrawal, donor-specific anti-HLA antibodies and donor-derived cell-free DNA were monitored before withdrawal and at follow-up. SARS-CoV-2 specific antibodies significantly increased in kidney transplant recipients after additional COVID-19 vaccination. The effect was most pronounced in individuals in whom mycophenolic acid was withdrawn during vaccination. Higher SARS-CoV-2 specific antibody titers were associated with better neutralization of SARS-CoV-2 delta and omicron variants. In patients with short-term withdrawal of mycophenolic acid, graft function and donor-derived cell-free DNA remained stable. No acute rejection episode occurred during short-term follow-up. However, resurgence of prior anti-HLA donor-specific antibodies was detected in 7 patients.

Neutralizing antibody response against the B.1.617.2 (delta) and the B.1.1.529 (omicron) variants after a third mRNA SARS-CoV-2 vaccine dose in kidney transplant recipients.

Seroconversion after COVID-19 vaccination is impaired in kidney transplant recipients. Emerging variants of concern such as the B.1.617.2 (delta) and the B.1.1.529 (omicron) variants pose an increasing threat to these patients. In this observational cohort study, we measured anti-S1 IgG, surrogate neutralizing, and anti-receptor-binding domain antibodies three weeks after a third mRNA vaccine dose in 49 kidney transplant recipients and compared results to 25 age-matched healthy controls. In addition, vaccine-induced neutralization of SARS-CoV-2 wild-type, the B.1.617.2 (delta), and the B.1.1.529 (omicron) variants was assessed using a live-virus assay. After a third vaccine dose, anti-S1 IgG, surrogate neutralizing, and anti-receptor-binding domain antibodies were significantly lower in kidney transplant recipients compared to healthy controls. Only 29/49 (59%) sera of kidney transplant recipients contained neutralizing antibodies against the SARS-CoV-2 wild-type or the B.1.617.2 (delta) variant and neutralization titers were significantly reduced compared to healthy controls (p < 0.001). Vaccine-induced cross-neutralization of the B.1.1.529 (omicron) variants was detectable in 15/35 (43%) kidney transplant recipients with seropositivity for anti-S1 IgG, surrogate neutralizing, and/or anti-RBD antibodies. Neutralization of the B.1.1.529 (omicron) variants was significantly reduced compared to neutralization of SARS-CoV-2 wild-type or the B.1.617.2 (delta) variant for both, kidney transplant recipients and healthy controls (p < .001 for all).

Neutralizing Antibody Activity Against the B.1.617.2 (delta) Variant Before and After a Third BNT162b2 Vaccine Dose in Hemodialysis Patients.

Hemodialysis patients are at high risk for severe COVID-19, and impaired seroconversion rates have been demonstrated after COVID-19 vaccination. Humoral immunity wanes over time and variants of concern with immune escape are posing an increasing threat. Little is known about protection against the B.1.617.2 (delta) variant of concern in hemodialysis patients before and after third vaccination. We determined anti-S1 IgG, surrogate neutralizing, and IgG antibodies against different SARS-CoV-2 epitopes in 84 hemodialysis patients directly before and three weeks after a third vaccine dose with BNT162b2. Third vaccination was performed after a median (IQR) of 119 (109-165) days after second vaccination. In addition, neutralizing activity against the B.1.617.2 (delta) variant was assessed in 31 seroconverted hemodialysis patients before and after third vaccination. Triple seropositivity for anti-S1 IgG, surrogate neutralizing, and anti-RBD antibodies increased from 31/84 (37%) dialysis patients after second to 80/84 (95%) after third vaccination. Neutralizing activity against the B.1.617.2 (delta) variant was significantly higher after third vaccination with a median (IQR) ID50 of 1:320 (1:160-1:1280) compared with 1:20 (0-1:40) before a third vaccine dose (P<0.001). The anti-S1 IgG index showed the strongest correlation with the ID50 against the B.1.617.2 (delta) variant determined by live virus neutralization (r=0.91). We demonstrate low neutralizing activity against the B.1.617.2 (delta) variant in dialysis patients four months after standard two-dose vaccination but a substantial increase after a third vaccine dose. Booster vaccination(s) should be considered earlier than 6 months after the second vaccine dose in immunocompromised individuals.

Contribution of autophagy machinery factors to HCV and SARS-CoV-2 replication organelle formation.

Positive-strand RNA viruses replicate in close association with rearranged intracellular membranes. For hepatitis C virus (HCV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), these rearrangements comprise endoplasmic reticulum (ER)-derived double membrane vesicles (DMVs) serving as RNA replication sites. Cellular factors involved in DMV biogenesis are poorly defined. Here, we show that despite structural similarity of viral DMVs with autophagosomes, conventional macroautophagy is dispensable for HCV and SARS-CoV-2 replication. However, both viruses exploit factors involved in autophagosome formation, most notably class III phosphatidylinositol 3-kinase (PI3K). As revealed with a biosensor, PI3K is activated in cells infected with either virus to produce phosphatidylinositol 3-phosphate (PI3P) while kinase complex inhibition or depletion profoundly reduces replication and viral DMV formation. The PI3P-binding protein DFCP1, recruited to omegasomes in early steps of autophagosome formation, participates in replication and DMV formation of both viruses. These results indicate that phylogenetically unrelated HCV and SARS-CoV-2 exploit similar components of the autophagy machinery to create their replication organelles.

Challenges for Targeting SARS-CoV-2 Proteases as a Therapeutic Strategy for COVID-19.

Two proteases produced by the SARS-CoV-2 virus, the main protease and papain-like protease, are essential for viral replication and have become the focus of drug development programs for treatment of COVID-19. We screened a highly focused library of compounds containing covalent warheads designed to target cysteine proteases to identify new lead scaffolds for both Mpro and PLpro proteases. These efforts identified a small number of hits for the Mpro protease and no viable hits for the PLpro protease. Of the Mpro hits identified as inhibitors of the purified recombinant protease, only two compounds inhibited viral infectivity in cellular infection assays. However, we observed a substantial drop in antiviral potency upon expression of TMPRSS2, a transmembrane serine protease that acts in an alternative viral entry pathway to the lysosomal cathepsins. This loss of potency is explained by the fact that our lead Mpro inhibitors are also potent inhibitors of host cell cysteine cathepsins. To determine if this is a general property of Mpro inhibitors, we evaluated several recently reported compounds and found that they are also effective inhibitors of purified human cathepsins L and B and showed similar loss in activity in cells expressing TMPRSS2. Our results highlight the challenges of targeting Mpro and PLpro proteases and demonstrate the need to carefully assess selectivity of SARS-CoV-2 protease inhibitors to prevent clinical advancement of compounds that function through inhibition of a redundant viral entry pathway.

Antiviral drug screen identifies DNA-damage response inhibitor as potent blocker of SARS-CoV-2 replication.

SARS-CoV-2 has currently precipitated the COVID-19 global health crisis. We developed a medium-throughput drug-screening system and identified a small-molecule library of 34 of 430 protein kinase inhibitors that were capable of inhibiting the SARS-CoV-2 cytopathic effect in human epithelial cells. These drug inhibitors are in various stages of clinical trials. We detected key proteins involved in cellular signaling pathways mTOR-PI3K-AKT, ABL-BCR/MAPK, and DNA-damage response that are critical for SARS-CoV-2 infection. A drug-protein interaction-based secondary screen confirmed compounds, such as the ATR kinase inhibitor berzosertib and torin2 with anti-SARS-CoV-2 activity. Berzosertib exhibited potent antiviral activity against SARS-CoV-2 in multiple cell types and blocked replication at the post-entry step. Berzosertib inhibited replication of SARS-CoV-1 and the Middle East respiratory syndrome coronavirus (MERS-CoV) as well. Our study highlights key promising kinase inhibitors to constrain coronavirus replication as a host-directed therapy in the treatment of COVID-19 and beyond as well as provides an important mechanism of host-pathogen interactions.

SARS-CoV-2 infection remodels the host protein thermal stability landscape.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a global threat to human health and has compromised economic stability. In addition to the development of an effective vaccine, it is imperative to understand how SARS-CoV-2 hijacks host cellular machineries on a system-wide scale so that potential host-directed therapies can be developed. In situ proteome-wide abundance and thermal stability measurements using thermal proteome profiling (TPP) can inform on global changes in protein activity. Here we adapted TPP to high biosafety conditions amenable to SARS-CoV-2 handling. We discovered pronounced temporal alterations in host protein thermostability during infection, which converged on cellular processes including cell cycle, microtubule and RNA splicing regulation. Pharmacological inhibition of host proteins displaying altered thermal stability or abundance during infection suppressed SARS-CoV-2 replication. Overall, this work serves as a framework for expanding TPP workflows to globally important human pathogens that require high biosafety containment and provides deeper resolution into the molecular changes induced by SARS-CoV-2 infection.