Suramin binds and inhibits infection of SARS-CoV-2 through both spike protein-heparan sulfate and ACE2 receptor interactions.

SARS-CoV-2 receptor binding domains (RBDs) interact with both the ACE2 receptor and heparan sulfate on the surface of host cells to enhance SARS-CoV-2 infection. We show that suramin, a polysulfated synthetic drug, binds to the ACE2 receptor and heparan sulfate binding sites on the RBDs of wild-type, Delta, and Omicron variants. Specifically, heparan sulfate and suramin had enhanced preferential binding for Omicron RBD, and suramin is most potent against the live SARS-CoV-2 Omicron variant (B.1.1.529) when compared to wild type and Delta (B.1.617.2) variants in vitro. These results suggest that inhibition of live virus infection occurs through dual SARS-CoV-2 targets of S-protein binding and previously reported RNA-dependent RNA polymerase inhibition and offers the possibility for this and other polysulfated molecules to be used as potential therapeutic and prophylactic options against COVID-19.

Cynomolgus Macaque Model for COVID-19 Delta Variant.

With the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants, which are randomly mutated, the dominant strains in regions are changing globally. The development of preclinical animal models is imperative to validate vaccines and therapeutics against SARS-CoV-2 variants. The objective of this study was to develop a non-human primate (NHP) model for SARS-CoV-2 Delta variant infection. Cynomolgus macaques infected with Delta variants showed infectious viruses and viral RNA in the upper (nasal and throat) and lower respiratory (lung) tracts during the acute phase of infection. After 3 days of infection, lesions consistent with diffuse alveolar damage were observed in the lungs. For cellular immune responses, all macaques displayed transient lymphopenia and neutrophilia in the early stages of infection. SARS-CoV-2 Delta variant spike protein-specific IgM, IgG, and IgA levels were significantly increased in the plasma of these animals 14 days after infection. This new NHP Delta variant infection model can be used for comparative analysis of the difference in severity between SARS-CoV-2 variants of concern and may be useful in the efficacy evaluation of vaccines and universal therapeutic drugs for mutations.

The Chimeric Adenovirus (Ad5/35) Expressing Engineered Spike Protein Confers Immunity against SARS-CoV-2 in Mice and Non-Human Primates.

Several COVID-19 platforms have been licensed across the world thus far, but vaccine platform research that can lead to effective antigen delivery is still ongoing. Here, we constructed AdCLD-CoV19 that could modulate humoral immunity by harboring SARS-CoV-2 antigens onto a chimeric adenovirus 5/35 platform that was effective in cellular immunity. By replacing the S1/S2 furin cleavage sequence of the SARS-CoV-2 Spike (S) protein mounted on AdCLD-CoV19 with the linker sequence, high antigen expression was confirmed in various cell lines. The high levels of antigen expression contributed to antigen-specific antibody activity in mice and non-human primates (NHPs) with a single vaccination of AdCLD-CoV19. Furthermore, the adenovirus-induced Th1 immune response was specifically raised for the S protein, and these immune responses protected the NHP against live viruses. While AdCLD-CoV19 maintained neutralizing antibody activity against various SARS-CoV-2 variants, it was reduced to single vaccination for ß and ο variants, and the reduced neutralizing antibody activity was restored with booster shots. Hence, AdCLD-CoV19 can prevent SARS-CoV-2 with a single vaccination, and the new vaccine administration strategy that responds to various variants can maintain the efficacy of the vaccine.

Germinal Center-Induced Immunity Is Correlated With Protection Against SARS-CoV-2 Reinfection But Not Lung Damage.

Germinal centers (GCs) elicit protective humoral immunity through a combination of antibody-secreting cells and memory B cells, following pathogen invasion or vaccination. However, the possibility of a GC response inducing protective immunity against reinfection following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remains unknown. We found GC activity was consistent with seroconversion observed in recovered macaques and humans. Rechallenge with a different clade of virus resulted in significant reduction in replicating virus titers in respiratory tracts in macaques with high GC activity. However, diffuse alveolar damage and increased fibrotic tissue were observed in lungs of reinfected macaques. Our study highlights the importance of GCs developed during natural SARS-CoV-2 infection in managing viral loads in subsequent infections. However, their ability to alleviate lung damage remains to be determined. These results may improve understanding of SARS-CoV-2-induced immune responses, resulting in better coronavirus disease 2019 (COVID-19) diagnosis, treatment, and vaccine development.

Host- and Species-Dependent Quasispecies Divergence of Severe Acute Respiratory Syndrome Coronavirus-2 in Non-human Primate Models.

Recently, newly emerging variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been continuously reported worldwide. However, the precise evaluation of SARS-CoV-2 microevolution in host is very limited because the exact genetic information of infected virus could not be acquired in human researches. In this report, we performed deep sequencing for seed virus and SARS-CoV-2 isolated in eight cynomolgus and rhesus macaques at 3 days postinoculation and evaluated single-nucleotide polymorphisms (SNPs) in SARS-CoV-2 by variant analysis. A total of 69 single-nucleotide variants (SNVs) were present in the 5'-untranslated region (UTR), 3'-UTR, ORF1ab, S, ORF3a, ORF8, and N genes of the seed virus passaged in VERO cells. Between those present on the seed virus and those on each SARS-CoV-2 isolated from the lungs of the macaques, a total of 29 variants was identified in 4 coding proteins (ORF1ab, S, ORF3a, and N) and non-coding regions (5'- and 3'-UTR). Variant number was significantly different according to individuals and ranged from 2 to 11. Moreover, the average major frequency variation was identified in six sites between the cynomolgus monkeys and rhesus macaques. As with diverse SNPs in SARS-CoV-2, the values of viral titers in lungs were significantly different according to individuals and species. Our study first revealed that the genomes of SARS-CoV-2 differ according to individuals and species despite infection of the identical virus in non-human primates (NHPs). These results are important for the interpretation of longitudinal studies evaluating the evolution of the SARS-CoV-2 in human beings and development of new diagnostics, vaccine, and therapeutics targeting SARS-CoV-2.

Immunization with RBD-P2 and N protects against SARS-CoV-2 in nonhuman primates.

Since the emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), various vaccines are being developed, with most vaccine candidates focusing on the viral spike protein. Here, we developed a previously unknown subunit vaccine comprising the receptor binding domain (RBD) of the spike protein fused with the tetanus toxoid epitope P2 (RBD-P2) and tested its efficacy in rodents and nonhuman primates (NHPs). We also investigated whether the SARS-CoV-2 nucleocapsid protein (N) could increase vaccine efficacy. Immunization with N and RBD-P2 (RBDP2/N) + alum increased T cell responses in mice and neutralizing antibody levels in rats compared with those obtained using RBD-P2 + alum. Furthermore, in NHPs, RBD-P2/N + alum induced slightly faster SARS-CoV-2 clearance than that induced by RBD-P2 + alum, albeit without statistical significance. Our study supports further development of RBD-P2 as a vaccine candidate against SARS-CoV-2. Also, it provides insights regarding the use of N in protein-based vaccines against SARS-CoV-2.

Soluble Spike DNA Vaccine Provides Long-Term Protective Immunity against SARS-CoV-2 in Mice and Nonhuman Primates.

The unprecedented and rapid spread of SARS-CoV-2 (severe acute respiratory syndrome-coronavirus-2) has motivated the need for a rapidly producible and scalable vaccine. Here, we developed a synthetic soluble SARS-CoV-2 spike (S) DNA-based vaccine candidate, GX-19. In mice, immunization with GX-19 elicited not only S-specific systemic and pulmonary antibody responses but also Th1-biased T cell responses in a dose-dependent manner. GX-19-vaccinated nonhuman primates seroconverted rapidly and exhibited a detectable neutralizing antibody response as well as multifunctional CD4+ and CD8+ T cell responses. Notably, when the immunized nonhuman primates were challenged at 10 weeks after the last vaccination with GX-19, they had reduced viral loads in contrast to non-vaccinated primates as a control. These findings indicate that GX-19 vaccination provides a durable protective immune response and also support further development of GX-19 as a vaccine candidate for SARS-CoV-2.

A therapeutic neutralizing antibody targeting receptor binding domain of SARS-CoV-2 spike protein.

Vaccines and therapeutics are urgently needed for the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we screen human monoclonal antibodies (mAb) targeting the receptor binding domain (RBD) of the viral spike protein via antibody library constructed from peripheral blood mononuclear cells of a convalescent patient. The CT-P59 mAb potently neutralizes SARS-CoV-2 isolates including the D614G variant without antibody-dependent enhancement effect. Complex crystal structure of CT-P59 Fab/RBD shows that CT-P59 blocks interaction regions of RBD for angiotensin converting enzyme 2 (ACE2) receptor with an orientation that is notably different from previously reported RBD-targeting mAbs. Furthermore, therapeutic effects of CT-P59 are evaluated in three animal models (ferret, hamster, and rhesus monkey), demonstrating a substantial reduction in viral titer along with alleviation of clinical symptoms. Therefore, CT-P59 may be a promising therapeutic candidate for COVID-19.

Transient Lymphopenia and Interstitial Pneumonia With Endotheliitis in SARS-CoV-2-Infected Macaques.

Using a reliable primate model is critical for developing therapeutic advances to treat humans infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Here, we exposed macaques to high titers of SARS-CoV-2 via combined transmission routes. We observed acute interstitial pneumonia with endotheliitis in the lungs of all infected macaques. All macaques had a significant loss of total lymphocytes during infection, which were restored over time. These data show that SARS-CoV-2 causes a coronavirus disease 2019 (COVID-19)-like disease in macaques. This new model could investigate the interaction between SARS-CoV-2 and the immune system to test therapeutic strategies.