Large-Scale Virtual Screening for the Discovery of SARS-CoV-2 Papain-like Protease (PLpro) Non-covalent Inhibitors.

The rapid global spread of the SARS-CoV-2 virus facilitated the development of novel direct-acting antiviral agents (DAAs). The papain-like protease (PLpro) has been proposed as one of the major SARS-CoV-2 targets for DAAs due to its dual role in processing viral proteins and facilitating the host's immune suppression. This dual role makes identifying small molecules that can effectively neutralize SARS-CoV-2 PLpro activity a high-priority task. However, PLpro drug discovery faces a significant challenge due to the high mobility and induced-fit effects in the protease's active site. Herein, we virtually screened the ZINC20 database with Deep Docking (DD) to identify prospective noncovalent PLpro binders and combined ultra-large consensus docking with two pharmacophore (ph4)-filtering strategies. The analysis of active compounds revealed their somewhat-limited diversity, likely attributed to the induced-fit nature of PLpro's active site in the crystal structures, and therefore, the use of rigid docking protocols poses inherited limitations. The top hits were assessed against recombinant viral proteins and live viruses, demonstrating desirable inhibitory activities. The best compound VPC-300195 (IC50: 15 µM) ranks among the top noncovalent PLpro inhibitors discovered through in silico methodologies. In the search for novel SARS-CoV-2 PLpro-specific chemotypes, the identified inhibitors could serve as diverse templates for the development of effective noncovalent PLpro inhibitors.

Reduced Magnitude and Durability of Humoral Immune Responses to COVID-19 mRNA Vaccines Among Older Adults.

BACKGROUND: The magnitude and durability of immune responses to coronavirus disease 2019 (COVID-19) mRNA vaccines remain incompletely characterized in the elderly. METHODS: Anti-spike receptor-binding domain (RBD) antibodies, angiotensin-converting enzyme 2 (ACE2) competition, and virus neutralizing activities were assessed in plasma from 151 health care workers and older adults (range, 24-98 years of age) 1 month following the first vaccine dose, and 1 and 3 months following the second dose. RESULTS: Older adults exhibited significantly weaker responses than younger health care workers for all humoral measures evaluated and at all time points tested, except for ACE2 competition activity after 1 vaccine dose. Moreover, older age remained independently associated with weaker responses even after correction for sociodemographic factors, chronic health condition burden, and vaccine-related variables. By 3 months after the second dose, all humoral responses had declined significantly in all participants, and remained significantly lower among older adults, who also displayed reduced binding antibodies and ACE2 competition activity towards the Delta variant. CONCLUSIONS: Humoral responses to COVID-19 mRNA vaccines are significantly weaker in older adults, and antibody-mediated activities in plasma decline universally over time. Older adults may thus remain at elevated risk of infection despite vaccination.

Older Adults Mount Less Durable Humoral Responses to Two Doses of COVID-19 mRNA Vaccine but Strong Initial Responses to a Third Dose.

BACKGROUND: Third coronavirus disease 2019 (COVID-19) vaccine doses are broadly recommended, but immunogenicity data remain limited, particularly in older adults. METHODS: We measured circulating antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein receptor-binding domain, ACE2 displacement, and virus neutralization against ancestral and omicron (BA.1) strains from prevaccine up to 1 month following the third dose, in 151 adults aged 24-98 years who received COVID-19 mRNA vaccines. RESULTS: Following 2 vaccine doses, humoral immunity was weaker, less functional, and less durable in older adults, where a higher number of chronic health conditions was a key correlate of weaker responses and poorer durability. One month after the third dose, antibody concentrations and function exceeded post-second-dose levels, and responses in older adults were comparable in magnitude to those in younger adults at this time. Humoral responses against omicron were universally weaker than against the ancestral strain after both the second and third doses. Nevertheless, after 3 doses, anti-omicron responses in older adults reached equivalence to those in younger adults. One month after 3 vaccine doses, the number of chronic health conditions, but not age, was the strongest consistent correlate of weaker humoral responses. CONCLUSIONS: Results underscore the immune benefits of third COVID-19 vaccine doses, particularly in older adults.

Synthetic Neoglycoconjugates of Hepta- and Nonamannoside Ligands for Eliciting Oligomannose-Specific HIV-1-Neutralizing Antibodies.

Oligomannose-type glycans on the spike protein of HIV-1 constitute relevant epitopes to elicit broadly neutralizing antibodies (bnAbs). Herein we describe an improved synthesis of α- and ß-linked hepta- and nonamannosyl ligands that were subsequently converted into BSA and CRM197 neoglycoconjugates. We assembled the ligands from anomeric 3-azidopropyl spacer glycosides from select 3-O-protected thiocresyl mannoside donors. Chain extensions were achieved using [4+3] or [4+5] block synthesis of thiocresyl and trichloroacetimidate glycosyl donors. Subsequent global deprotection generated the 3-aminopropyl oligosaccharide ligands. ELISA binding data obtained with the ß-anomeric hepta- and nonamannosyl conjugates with a selection of HIV-1 bnAbs showed comparable binding of both mannosyl ligands by Fab fragments yet lesser binding of the nonasaccharide conjugate by the corresponding IgG antibodies. These results support previous observations that a complete Man9 structure might not be the preferred antigenic binding motif for some oligomannose-specific antibodies, and have implications for glycoside designs to elicit oligomannose-targeted HIV-1-neutralizing antibodies.

Older Adults Mount Less Durable Humoral Responses to a Two-dose COVID-19 mRNA Vaccine Regimen, but Strong Initial Responses to a Third Dose.

BACKGROUND: Third COVID-19 vaccine doses are broadly recommended, but immunogenicity data remain limited, particularly in older adults. METHODS: We measured circulating antibodies against the SARS-CoV-2 spike protein receptor-binding domain, ACE2 displacement, and virus neutralization against ancestral and Omicron (BA.1) strains from pre-vaccine up to one month following the third dose, in 151 adults aged 24-98 years who received COVID-19 mRNA vaccines. RESULTS: Following two vaccine doses, humoral immunity was weaker, less functional and less durable in older adults, where a higher number of chronic health conditions was a key correlate of weaker responses and poorer durability. Third doses boosted antibody binding and function to higher levels than second-doses, and induced responses in older adults that were comparable in magnitude to those in younger adults. Humoral responses against Omicron were universally weaker than against the ancestral strain after both second and third doses; nevertheless, after three doses, anti-Omicron responses in older adults reached equivalence to those in younger adults. After three vaccine doses, the number of chronic health conditions, but not age per se, was the strongest consistent correlate of weaker humoral responses. CONCLUSION: Results underscore the immune benefits of third COVID-19 vaccine doses, particularly in older adults.

Humoral immune responses to COVID-19 vaccination in people living with HIV receiving suppressive antiretroviral therapy.

Humoral responses to COVID-19 vaccines in people living with HIV (PLWH) remain incompletely characterized. We measured circulating antibodies against the SARS-CoV-2 spike protein receptor-binding domain (RBD), ACE2 displacement and viral neutralization activities one month following the first and second COVID-19 vaccine doses, and again 3 months following the second dose, in 100 adult PLWH and 152 controls. All PLWH were receiving suppressive antiretroviral therapy, with median CD4+ T-cell counts of 710 (IQR 525-935) cells/mm3, though nadir CD4+ T-cell counts ranged as low as <10 cells/mm3. After adjustment for sociodemographic, health and vaccine-related variables, HIV infection was associated with lower anti-RBD antibody concentrations and ACE2 displacement activity after one vaccine dose. Following two doses however, HIV was not significantly associated with the magnitude of any humoral response after multivariable adjustment. Rather, older age, a higher burden of chronic health conditions, and dual ChAdOx1 vaccination were associated with lower responses after two vaccine doses. No significant correlation was observed between recent or nadir CD4+ T-cell counts and responses to two vaccine doses in PLWH. These results indicate that PLWH with well-controlled viral loads and CD4+ T-cell counts in a healthy range generally mount strong initial humoral responses to dual COVID-19 vaccination. Factors including age, co-morbidities, vaccine brand, response durability and the rise of new SARS-CoV-2 variants will influence when PLWH will benefit from additional doses. Further studies of PLWH who are not receiving antiretroviral treatment or who have low CD4+ T-cell counts are needed, as are longer-term assessments of response durability.

Weak humoral immune reactivity among residents of long-term care facilities following one dose of the BNT162b2 mRNA COVID-19 vaccine.

Background: Several Canadian provinces are extending the interval between COVID-19 vaccine doses to increase population vaccine coverage more rapidly. However, immunogenicity of these vaccines after one dose is incompletely characterized, particularly among the elderly, who are at greatest risk of severe COVID-19. Methods: We assessed SARS-CoV-2 humoral responses pre-vaccine and one month following the first dose of BNT162b2 mRNA vaccine, in 12 COVID-19 seronegative residents of long-term care facilities (median age, 82 years), 18 seronegative healthcare workers (HCW; median age, 36 years) and 4 convalescent HCW. Total antibody responses to SARS-CoV-2 nucleocapsid (N) and spike protein receptor binding domain (S/RBD) were assessed using commercial immunoassays. We quantified IgG and IgM responses to S/RBD and determined the ability of antibodies to block S/RBD binding to ACE2 receptor using ELISA. Neutralizing antibody activity was also assessed using pseudovirus and live SARS-CoV-2. Results: After one vaccine dose, binding antibodies against S/RBD were ~4-fold lower in residents compared to HCW (p<0.001). Inhibition of ACE2 binding was 3-fold lower in residents compared to HCW (p=0.01) and pseudovirus neutralizing activity was 2-fold lower (p=0.003). While six (33%) seronegative HCW neutralized live SARS-CoV-2, only one (8%) resident did (p=0.19). In contrast, convalescent HCW displayed 7- to 20-fold higher levels of binding antibodies and substantial ability to neutralize live virus after one dose. Interpretation: Extending the interval between COVID-19 vaccine doses may pose a risk to the elderly due to lower vaccine immunogenicity in this group. We recommend that second doses not be delayed in elderly individuals.

Humoral immune responses to COVID-19 vaccination in people living with HIV receiving suppressive antiretroviral therapy.

Humoral responses to COVID-19 vaccines in people living with HIV (PLWH) remain incompletely understood. We measured circulating antibodies against the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein, ACE2 displacement and live viral neutralization activities one month following the first and second COVID-19 vaccine doses in 100 adult PLWH and 152 controls. All PLWH were receiving suppressive antiretroviral therapy, with median CD4+ T-cell counts of 710 (IQR 525-935) cells/mm 3 . Nadir CD4+ T-cell counts ranged as low as <10 (median 280; IQR 120-490) cells/mm 3 . After adjustment for sociodemographic, health and vaccine-related variables, HIV infection was significantly associated with 0.2 log 10 lower anti-RBD antibody concentrations (p=0.03) and ∻11% lower ACE2 displacement activity (p=0.02), but not lower viral neutralization (p=0.1) after one vaccine dose. Following two doses however, HIV was no longer significantly associated with the magnitude of any response measured. Rather, older age, a higher burden of chronic health conditions, and having received two ChAdOx1 doses (versus a heterologous or dual mRNA vaccine regimen) were independently associated with lower responses. After two vaccine doses, no significant correlation was observed between the most recent or nadir CD4+ T-cell counts and vaccine responses in PLWH. These results suggest that PLWH with well-controlled viral loads on antiretroviral therapy and CD4+ T-cell counts in a healthy range will generally not require a third COVID-19 vaccine dose as part of their initial immunization series, though other factors such as older age, co-morbidities, vaccine regimen type, and durability of vaccine responses will influence when this group may benefit from additional doses. Further studies of PLWH who are not receiving antiretroviral treatment and/or who have low CD4+ T-cell counts are needed.

Responses of human mast cells and epithelial cells following exposure to influenza A virus.

As a part of innate immune defense, the role of mast cells during viral replication has been incompletely understood. In this study, we characterized and compared the responses of the human mast cell line, LAD2, and human lung epithelial cell line, Calu-3, against three influenza A virus strains; A/PR/8/34 (H1N1), A/WS/33 (H1N1) and A/HK/8/68 (H3N2). We found that there were strain-dependent mast cell responses, and different profiles of cytokine, chemokine and antiviral gene expression between the two cell types. All three strains did not induce histamine or ß-hexosaminidase release in LAD2. A/HK/8/68 induced release of prostaglandin D2 in LAD2, whereas A/PR/8/34 and A/WS/33 did not. We found that, among those examined, only CCL4 (by A/PR/8/34) was statistically significantly released from LAD2 cells. Furthermore, there was increased mRNA expression of viral recognition receptors (RIG-I and MDA5) and antiviral protein, viperin, but levels and kinetics of the expression were different among the cell types, as well as by the strains examined. Our findings highlight the variability in innate response to different strains of influenza A virus in two human cell types, indicating that further investigation is needed to understand better the role of mast cells and epithelial cells in innate immunity against influenza A viruses.