A Second-Generation Oral SARS-CoV-2 Main Protease Inhibitor Clinical Candidate for the Treatment of COVID-19.

Despite the record-breaking discovery, development and approval of vaccines and antiviral therapeutics such as Paxlovid, coronavirus disease 2019 (COVID-19) remained the fourth leading cause of death in the world and third highest in the United States in 2022. Here, we report the discovery and characterization of PF-07817883, a second-generation, orally bioavailable, SARS-CoV-2 main protease inhibitor with improved metabolic stability versus nirmatrelvir, the antiviral component of the ritonavir-boosted therapy Paxlovid. We demonstrate the in vitro pan-human coronavirus antiviral activity and off-target selectivity profile of PF-07817883. PF-07817883 also demonstrated oral efficacy in a mouse-adapted SARS-CoV-2 model at plasma concentrations equivalent to nirmatrelvir. The preclinical in vivo pharmacokinetics and metabolism studies in human matrices are suggestive of improved oral pharmacokinetics for PF-07817883 in humans, relative to nirmatrelvir. In vitro inhibition/induction studies against major human drug metabolizing enzymes/transporters suggest a low potential for perpetrator drug-drug interactions upon single-agent use of PF-07817883.

Generation of a VeroE6 Pgp gene knock out cell line and its use in SARS-CoV-2 antiviral study.

Vero cells are widely used for antiviral tests and virology research for SARS-CoV-2 as well as viruses from various other families. However, Vero cells generally express high levels of multi-drug resistance 1 (MDR1) or Pgp protein, the efflux transporter of foreign substances including many antiviral compounds, affecting the antiviral activity as well as interpretation of data. To address this, a Pgp gene knockout VeroE6 cell line (VeroE6-Pgp-KO) was generated using CRISPR-CAS9 technology. These cells no longer expressed the Pgp protein as indicated by flow cytometry analysis following staining with a Pgp-specific monoclonal antibody. They also showed significantly reduced efflux transporter activity in the calcein acetoxymethyl ester (calcein AM) assay. The VeroE6-Pgp-KO cells and the parental VeroE6 cells were each infected with SARS-CoV-2 to test antiviral activities of remdesivir and nirmatrelvir, two known Pgp substrates, in the presence or absence of a Pgp inhibitor. The compounds showed antiviral activities in VeroE6-Pgp-KO cells similar to that observed in the presence of the Pgp inhibitor. Thus, the newly established VeroE6-Pgp-KO cell line adds a new in vitro virus infection system for SARS-CoV-2 and possibly other viruses to test antiviral therapies without a need to control the Pgp activity. Removal of the Pgp inhibitor for antiviral assays will lead to less data variation and prevent failed assays.

Neutralizing Antibodies to Human Cytomegalovirus Recombinant Proteins Reduce Infection in an Ex Vivo Model of Developing Human Placentas.

Human cytomegalovirus (HCMV) is the leading viral cause of congenital disease and permanent birth defects worldwide. Although the development of an effective vaccine is a public health priority, no vaccines are approved. Among the major antigenic targets are glycoproteins in the virion envelope, including gB, which facilitates cellular entry, and the pentameric complex (gH/gL/pUL128-131), required for the infection of specialized cell types. In this study, sera from rabbits immunized with the recombinant pentameric complex were tested for their ability to neutralize infection of epithelial cells, fibroblasts, and primary placental cell types. Sera from rhesus macaques immunized with recombinant gB or gB plus pentameric complex were tested for HCMV neutralizing activity on both cultured cells and cell column cytotrophoblasts in first-trimester chorionic villus explants. Sera from rabbits immunized with the pentameric complex potently blocked infection by pathogenic viral strains in amniotic epithelial cells and cytotrophoblasts but were less effective in fibroblasts and trophoblast progenitor cells. Sera from rhesus macaques immunized with the pentameric complex and gB more strongly reduced infection in fibroblasts, epithelial cells, and chorionic villus explants than sera from immunization with gB alone. These results suggest that the pentameric complex and gB together elicit antibodies that could have potential as prophylactic vaccine antigens.

Horizontal Transmission of Cytomegalovirus in a Rhesus Model Despite High-Level, Vaccine-Elicited Neutralizing Antibody and T-Cell Responses.

The development of a vaccine to prevent congenital human cytomegalovirus (HCMV) disease is a public health priority. We tested rhesus CMV (RhCMV) prototypes of HCMV vaccine candidates in a seronegative macaque oral challenge model. Immunogens included a recombinant pentameric complex (PC; gH/gL/pUL128/pUL130/pUL131A), a postfusion gB ectodomain, and a DNA plasmid that encodes pp65-2. Immunization with QS21-adjuvanted PC alone or with the other immunogens elicited neutralizing titers comparable to those elicited by RhCMV infection. Similarly, immunization with all 3 immunogens elicited pp65-specific cytotoxic T-cell responses comparable to those elicited by RhCMV infection. RhCMV readily infected immunized animals and was detected in saliva, blood, and urine after challenge in quantities similar to those in placebo-immunized animals. If HCMV evades vaccine-elicited immunity in humans as RhCMV evaded immunity in macaques, a HCMV vaccine must elicit immunity superior to, or different from, that elicited by the prototype RhCMV vaccine to block horizontal transmission.

A novel approach to generate a recombinant toxoid vaccine against Clostridium difficile.

The Clostridium difficile toxins A and B are primarily responsible for symptoms of C. difficile associated disease and are prime targets for vaccine development. We describe a plasmid-based system for the production of genetically modified toxins in a non-sporulating strain of C. difficile that lacks the toxin genes tcdA and tcdB. TcdA and TcdB mutations targeting established glucosyltransferase cytotoxicity determinants were introduced into recombinant plasmids and episomally expressed toxin mutants purified from C. difficile transformants. TcdA and TcdB mutants lacking glucosyltransferase and autoproteolytic processing activities were ~10 000-fold less toxic to cultured human IMR-90 cells than corresponding recombinant or native toxins. However, both mutants retained residual cytotoxicity that could be prevented by preincubating the antigens with specific antibodies or by formalin treatment. Such non-toxic formalin-treated mutant antigens were immunogenic and protective in a hamster model of infection. The remaining toxicity of untreated TcdA and TcdB mutant antigens was associated with cellular swelling, a phenotype consistent with pore-induced membrane leakage. TcdB substitution mutations previously shown to block vesicular pore formation and toxin translocation substantially reduced residual toxicity. We discuss the implications of these results for the development of a C. difficile toxoid vaccine.