AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

Broadly Protective Neuraminidase-Based Influenza Vaccines and Monoclonal Antibodies: Target Epitopes and Mechanisms of Action

Neuraminidase (NA) is an important surface protein on influenza virions, playing an essential role in the viral life cycle and being a key target of the immune system. Despite the importance of NA-based immunity, current vaccines are focused on the hemagglutinin (HA) protein as the target for protective antibodies, and the amount of NA is not standardized in virion-based vaccines. Antibodies targeting NA are predominantly protective, reducing infection severity and viral shedding. Recently, NA-specific monoclonal antibodies have been characterized, and their target epitopes have been identified. This review summarizes the characteristics of NA, NA-specific antibodies, the mechanism of NA inhibition, and the recent efforts towards developing NA-based and NA-incorporating influenza vaccines

Antibody recognition of the Pneumovirus fusion protein trimer interface.

Human metapneumovirus (hMPV) is a leading cause of viral respiratory infection in children, and can cause severe lower respiratory tract infection in infants, the elderly, and immunocompromised patients. However, there remain no licensed vaccines or specific treatments for hMPV infection. Although the hMPV fusion (F) protein is the sole target of neutralizing antibodies, the immunological properties of hMPV F remain poorly understood. To further define the humoral immune response to the hMPV F protein, we isolated two new human monoclonal antibodies (mAbs), MPV458 and MPV465. Both mAbs are neutralizing in vitro and were determined to target a unique antigenic site using competitive biolayer interferometry. We determined both MPV458 and MPV465 have higher affinity for monomeric hMPV F than trimeric hMPV F. MPV458 was co-crystallized with hMPV F, and the mAb primarily interacts with an alpha helix on the F2 region of the hMPV F protein. Surprisingly, the major epitope for MPV458 lies within the trimeric interface of the hMPV F protein, suggesting significant breathing of the hMPV F protein must occur for host immune recognition of the novel epitope. In addition, significant glycan interactions were observed with a somatically mutated light chain framework residue. The data presented identifies a novel epitope on the hMPV F protein for epitope-based vaccine design, and illustrates a new mechanism for human antibody neutralization of viral glycoproteins

Pneumocystis Pneumonia: Immunity, Vaccines, and Treatments

For individuals who are immunocompromised, the opportunistic fungal pathogen Pneumocystis jirovecii is capable of causing life-threatening pneumonia as the causative agent of Pneumocystis pneumonia (PCP). PCP remains an acquired immunodeficiency disease (AIDS)-defining illness in the era of antiretroviral therapy. In addition, a rise in non-human immunodeficiency virus (HIV)-associated PCP has been observed due to increased usage of immunosuppressive and immunomodulating therapies. With the persistence of HIV-related PCP cases and associated morbidity and mortality, as well as difficult to diagnose non-HIV-related PCP cases, an improvement over current treatment and prevention standards is warranted. Current therapeutic strategies have primarily focused on the administration of trimethoprim-sulfamethoxazole, which is effective at disease prevention. However, current treatments are inadequate for treatment of PCP and prevention of PCP-related death, as evidenced by consistently high mortality rates for those hospitalized with PCP. There are no vaccines in clinical trials for the prevention of PCP, and significant obstacles exist that have slowed development, including host range specificity, and the inability to culture Pneumocystis spp. in vitro. In this review, we overview the immune response to Pneumocystis spp., and discuss current progress on novel vaccines and therapies currently in the preclinical and clinical pipeline

Adjuvant-Mediated Differences in Antibody Responses to Computationally Optimized Hemagglutinin and Neuraminidase Vaccines

Computationally optimized broadly reactive antigens (COBRAs) are a next-generation universal influenza vaccine candidate. However, how these COBRAs induce antibody responses when combined with different adjuvants has not previously been well-characterized. Therefore, we performed in vivo studies with an HA-based H1 COBRA, Y2, and an NA-based N1 COBRA, N1-I, to assess this effect for the H1N1 subtype. We tested the adjuvants AddaVax, AddaS03, CpG, and Alhydrogel. AddaS03 performed the best, eliciting high IgG titers and hemagglutination inhibition (HAI) activity for Y2 immunizations. Interestingly, serum antibody epitopes were relatively similar across adjuvant groups. Moreover, following N1-I immunization with these adjuvants, AddaS03 also elicited the highest IgG and neuraminidase inhibition (NAI) titers against the 2009 pandemic virus, A/California/07/2009 (A/CA/09). These results inform adjuvant selection efforts for H1 and N1 COBRA HA and NA antigens in a mouse model

Human Metapneumovirus (hMPV) Infection and MPV467 Treatment in Immunocompromised Cotton Rats <i>Sigmodon hispidus</i>

Human metapneumovirus (hMPV) is an important cause of respiratory disease in immunocompromised individuals, yet hMPV infection has not been modeled before in immunocompromised animals. In this work, cotton rats S. hispidus immunosuppressed by cyclophosphamide were infected with hMPV, and viral replication and pulmonary inflammation in these animals were compared to those in normal hMPV-infected S. hispidus. The efficacy of prophylactic and therapeutic administration of the anti-hMPV antibody MPV467 was also evaluated. Immunosuppressed animals had higher pulmonary and nasal titers of hMPV on day 5 post-infection compared to normal animals, and large amounts of hMPV were still present in the respiratory tract of immunosuppressed animals on days 7 and 9 post-infection, indicating prolonged viral replication. Immunosuppression was accompanied by reduced pulmonary histopathology in hMPV-infected cotton rats compared to normal animals; however, a delayed increase in pathology and pulmonary chemokine expression was seen in immunosuppressed cotton rats. Prophylactic and therapeutic MPV467 treatments protected both upper and lower respiratory tracts against hMPV infection. The lung pathology and pulmonary expression of IP-10 and MIP-1α mRNA were reduced by therapeutic MPV467 administration. These results indicate that immunosuppressed cotton rats represent a useful model for studying hMPV pathogenesis and for evaluating therapeutics that could alleviate hMPV-induced disease in immunocompromised subjects

Human Metapneumovirus (hMPV) Infection and MPV467 Treatment in Immunocompromised Cotton Rats Sigmodon hispidus

Human metapneumovirus (hMPV) is an important cause of respiratory disease in immunocompromised individuals, yet hMPV infection has not been modeled before in immunocompromised animals. In this work, cotton rats S. hispidus immunosuppressed by cyclophosphamide were infected with hMPV, and viral replication and pulmonary inflammation in these animals were compared to those in normal hMPV-infected S. hispidus. The efficacy of prophylactic and therapeutic administration of the anti-hMPV antibody MPV467 was also evaluated. Immunosuppressed animals had higher pulmonary and nasal titers of hMPV on day 5 post-infection compared to normal animals, and large amounts of hMPV were still present in the respiratory tract of immunosuppressed animals on days 7 and 9 post-infection, indicating prolonged viral replication. Immunosuppression was accompanied by reduced pulmonary histopathology in hMPV-infected cotton rats compared to normal animals; however, a delayed increase in pathology and pulmonary chemokine expression was seen in immunosuppressed cotton rats. Prophylactic and therapeutic MPV467 treatments protected both upper and lower respiratory tracts against hMPV infection. The lung pathology and pulmonary expression of IP-10 and MIP-1&alpha; mRNA were reduced by therapeutic MPV467 administration. These results indicate that immunosuppressed cotton rats represent a useful model for studying hMPV pathogenesis and for evaluating therapeutics that could alleviate hMPV-induced disease in immunocompromised subjects

A general computational design strategy for stabilizing viral class I fusion proteins

Abstract Many pathogenic viruses rely on class I fusion proteins to fuse their viral membrane with the host cell membrane. To drive the fusion process, class I fusion proteins undergo an irreversible conformational change from a metastable prefusion state to an energetically more stable postfusion state. Mounting evidence underscores that antibodies targeting the prefusion conformation are the most potent, making it a compelling vaccine candidate. Here, we establish a computational design protocol that stabilizes the prefusion state while destabilizing the postfusion conformation. With this protocol, we stabilize the fusion proteins of the RSV, hMPV, and SARS-CoV-2 viruses, testing fewer than a handful of designs. The solved structures of these designed proteins from all three viruses evidence the atomic accuracy of our approach. Furthermore, the humoral response of the redesigned RSV F protein compares to that of the recently approved vaccine in a mouse model. While the parallel design of two conformations allows the identification of energetically sub-optimal positions for one conformation, our protocol also reveals diverse molecular strategies for stabilization. Given the clinical significance of viruses using class I fusion proteins, our algorithm can substantially contribute to vaccine development by reducing the time and resources needed to optimize these immunogens

Structure, immunogenicity, and conformation-dependent receptor binding of the post-fusion human metapneumovirus F protein

Human metapneumovirus (hMPV) is an important cause of acute viral respiratory infection. As the only target of neutralizing antibodies, the hMPV fusion (F) protein has been a major focus for vaccine development and targeting by drugs and monoclonal antibodies (mAbs). While X-ray structures of trimeric pre-fusion and post-fusion hMPV F proteins from genotype A, and monomeric pre-fusion hMPV F protein from genotype B have been determined, structural data for the post-fusion conformation for genotype B is lacking. We determined the crystal structure of this protein and compared the structural differences of post-fusion hMPV F between hMPV A and B genotypes. We also assessed the receptor binding properties of the hMPV F protein to heparin and heparan sulfate (HS). A library of HS oligomers was used to verify the HS binding activity of hMPV F, and several compounds showed binding to predominantly pre-fusion hMPV F, but had limited binding to post-fusion hMPV F. Furthermore, mAbs to antigenic sites III and the 66-87 intratrimeric epitope block heparin binding. In addition, we evaluated the efficacy of post-fusion hMPV B2 F protein as a vaccine candidate in BALB/c mice. Mice immunized with hMPV B2 post-fusion F protein showed a balanced Th1/Th2 immune response and generated neutralizing antibodies against both subgroup A2 and B2 hMPV strains, which protected the mice from hMPV challenge. Antibody competition analysis revealed the antibodies generated by immunization target two known antigenic sites (III and IV) on the hMPV F protein. Overall, this study provides new characteristics of the hMPV F protein, which may be informative for vaccine and therapy development. Importance Human metapneumovirus (hMPV) is an important cause of viral respiratory disease. In this paper, we report the X-ray crystal structure of the hMPV fusion (F) protein in the post-fusion conformation from genotype B. We also assessed binding of the hMPV F protein to heparin and heparan sulfate, a previously reported receptor for the hMPV F protein. Furthermore, we determined the immunogenicity and protective efficacy of post-fusion hMPV B2 F protein, which is the first study using a homogenous conformation of the protein. Antibodies generated in response to vaccination give a balanced Th1/Th2 response and target two previously discovered neutralizing epitopes