Invention of MK-7845, a SARS-CoV-2 3CL Protease Inhibitor Employing a Novel Difluorinated Glutamine Mimic.

As SARS-CoV-2 continues to circulate, antiviral treatments are needed to complement vaccines. The virus's main protease, 3CLPro, is an attractive drug target in part because it recognizes a unique cleavage site, which features a glutamine residue at the P1 position and is not utilized by human proteases. Herein, we report the invention of MK-7845, a novel reversible covalent 3CLPro inhibitor. While most covalent inhibitors of SARS-CoV-2 3CLPro reported to date contain an amide as a Gln mimic at P1, MK-7845 bears a difluorobutyl substituent at this position. SAR analysis and X-ray crystallographic studies indicate that this group interacts with His163, the same residue that forms a hydrogen bond with the amide substituents typically found at P1. In addition to promising in vivo efficacy and an acceptable projected human dose with unboosted pharmacokinetics, MK-7845 exhibits favorable properties for both solubility and absorption that may be attributable to the unusual difluorobutyl substituent.

Evaluation of luciferase and prefusion-stabilized F protein from respiratory syncytial virus mRNA/LNPs in pre-clinical models using jet delivery compared to needle and syringe.

Described here is the evaluation of a luciferase (luc) and respiratory syncytial virus (RSV) messenger RNA / lipid nanoparticle (mRNA/LNP) vaccine using a Needle-free Injection System, Tropis®, from PharmaJet® (Golden, Colorado USA). Needle-free jet delivery offers an alternative to needle/syringe. To perform this assessment, compatibility studies with Tropis were first performed with a luc mRNA/LNP and compared to needle/syringe. Although minor changes in particle size and encapsulation efficiency were observed when using Tropis on the benchtop, in vitro luciferase activity remained the same. Next, the luc mRNA/LNP was administered to rats intramuscularly using Tropis or needle/syringe and tracking of the injection and distribution was performed. Lastly, an mRNA encoding a prefusion-stabilized F protein from RSV was delivered intramuscularly using both Tropis and needle/syringe at 1 and 5 mcg mRNA. An equivalent IgG response was observed using both Tropis and needle/syringe. The cell mediated immune (CMI) response was also evaluated, and responses to RSV-F were detected from animals immunized with needle/syringe at all dose levels, and from the animals immunized with Tropis in the 5 and 25 ug groups. These results indicated that delivery of mRNA/LNPs with Tropis is a potential means of administration and an alternative to needle/syringe.

Phage-derived anti-idiotype and anti-YTE antibodies in development of MK-1654 pharmacokinetic and immune response assays.

Background: MK-1654 is a fully human monoclonal antibody with YTE mutations currently in phase III clinical trials for prophylactic use in protecting infants from human respiratory syncytial virus infection. Materials & methods: We generated anti-idiotype (anti-ID) and anti-YTE antibodies against MK-1654 by panning with MorphoSys HuCal phage libraries, and used the antibodies in the development of MK-1654 pharmacokinetic (PK) and immune response (IR) assays. Results: Detection of MK-1654 in nonhuman primate and human nasal wash samples showed combined use of anti-ID and anti-YTE antibodies can deliver desired sensitivity and accuracy in PK studies. IR studies showed anti-ID can serve as suitable positive control in neutralizing antibody assays. Conclusion: Phage-derived anti-IDs and anti-YTEs are suitable for PK and IR assays.

Conserved allosteric inhibitory site on the respiratory syncytial virus and human metapneumovirus RNA-dependent RNA polymerases.

Respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) are related RNA viruses responsible for severe respiratory infections and resulting disease in infants, elderly, and immunocompromised adults1-3. Therapeutic small molecule inhibitors that bind to the RSV polymerase and inhibit viral replication are being developed, but their binding sites and molecular mechanisms of action remain largely unknown4. Here we report a conserved allosteric inhibitory site identified on the L polymerase proteins of RSV and HMPV that can be targeted by a dual-specificity, non-nucleoside inhibitor, termed MRK-1. Cryo-EM structures of the inhibitor in complexes with truncated RSV and full-length HMPV polymerase proteins provide a structural understanding of how MRK-1 is active against both viruses. Functional analyses indicate that MRK-1 inhibits conformational changes necessary for the polymerase to engage in RNA synthesis initiation and to transition into an elongation mode. Competition studies reveal that the MRK-1 binding pocket is distinct from that of a capping inhibitor with an overlapping resistance profile, suggesting that the polymerase conformation bound by MRK-1 may be distinct from that involved in mRNA capping. These findings should facilitate optimization of dual RSV and HMPV replication inhibitors and provide insights into the molecular mechanisms underlying their polymerase activities.

Split-Dose Administration Enhances Immune Responses Elicited by a mRNA/Lipid Nanoparticle Vaccine Expressing Respiratory Syncytial Virus F Protein.

mRNA vaccines have recently received significant attention due to their role in combating the SARS-CoV-2 pandemic. As a platform, mRNA vaccines have been shown to elicit strong humoral and cellular immune responses with acceptable safety profiles for prophylactic use. Despite their potential, industrial challenges have limited realization of the vaccine platform on a global scale. Critical among these challenges are supply chain considerations, including mRNA production, cost of goods, and vaccine frozen-chain distribution. Here, we assess the delivery of lipid nanoparticle-encapsulated mRNA (mRNA/LNP) vaccines using a split-dose immunization regimen as an approach to develop mRNA dose-sparing vaccine regimens with potential to mitigate mRNA supply chain challenges. Our data demonstrate that immunization by a mRNA/LNP vaccine encoding respiratory syncytial virus pre-F (RSV pre-F) over a 9 day period elicits comparable or superior magnitude of antibodies when compared to traditional bolus immunization of the vaccine. The split-dose immunization regimens evaluated in our studies were designed to mimic reported drug or antigen release profiles from microneedle patches, highlighting the potential benefit of pairing mRNA vaccines with patch-based delivery technologies to enable sustained release and solid-state stabilization. Overall, our findings provide a proof of concept to support further investigations into the development of sustained delivery approaches for mRNA/LNP vaccines.

Outer membrane protein complex as a carrier for malaria transmission blocking antigen Pfs230.

Malaria transmission blocking vaccines (TBV) target the mosquito stage of parasite development by passive immunization of mosquitoes feeding on a vaccinated human. Through uptake of vaccine-induced antibodies in a blood meal, mosquito infection is halted and hence transmission to another human host is blocked. Pfs230 is a gametocyte and gamete surface antigen currently under clinical evaluation as a TBV candidate. We have previously shown that chemical conjugation of poorly immunogenic TBV antigens to Exoprotein A (EPA) can enhance their immunogenicity. Here, we assessed Outer Membrane Protein Complex (OMPC), a membrane vesicle derived from Neisseria meningitidis, as a carrier for Pfs230. We prepared Pfs230-OMPC conjugates with varying levels of antigen load and examined immunogenicity in mice. Chemical conjugation of Pfs230 to OMPC enhanced immunogenicity and functional activity of the Pfs230 antigen, and OMPC conjugates achieved 2-fold to 20-fold higher antibody titers than Pfs230-EPA/AdjuPhos® at different doses. OMPC conjugates were highly immunogenic even at low doses, indicating a dose-sparing effect. EPA conjugates induced an IgG subclass profile biased towards a Th2 response, whereas OMPC conjugates induced a strong Th1-biased immune response with high levels of IgG2, which can benefit Pfs230 antibody functional activity, which depends on complement activation. OMPC is a promising carrier for Pfs230 vaccines.

Spectroscopic analysis of chlamydial major outer membrane protein in support of structure elucidation.

Chlamydial major outer membrane protein (MOMP) is the major protein constituent of the bacterial pathogen Chlamydia trachomatis. Chlamydia trachomatis Serovars D-K are the leading cause of genital tract infections which can lead to infertility or ectopic pregnancies. A vaccine against Chlamydia is highly desirable but currently not available. MOMP accounts for ~ 60% of the chlamydial protein mass and is considered to be one of the lead vaccine candidates against C. trachomatis. We report on the spectroscopic analysis of C. trachomatis native MOMP Serovars D, E, F, and J as well as C. muridarum MOMP by size exclusion chromatography multi angle light scattering (SEC MALS), circular dichroism (CD) and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). MOMP was purified from the native bacterium grown in either adherent HeLa cells or in different suspension cell lines. Our results confirm that MOMP forms homo-trimers in detergent micelles. The secondary structure composition of C. trachomatis MOMP was conserved across serovars, but different from composition of C. muridarum MOMP with a 13% (CD) to 18% (ATR-FTIR) reduction in ß-sheet conformation for C. trachomatis MOMP. When Serovar E MOMP was isolated from suspension cell lines the α-helix content increased by 7% (CD) to 13% (ATIR-FTIR). Maintenance of a native-like tertiary and quaternary structure in subunit vaccines is important for the generation of protective antibodies. This biophysical characterization of MOMP presented here serves, in the absence of functional assays, as a method for monitoring the structural integrity of MOMP.

Recombinant expression of Chlamydia trachomatis major outer membrane protein in E. Coli outer membrane as a substrate for vaccine research.

BACKGROUND: Chlamydia trachomatis is a human pathogen which causes a number of pathologies, including genital tract infections in women that can result in tubal infertility. Prevention of infection and disease control might be achieved through vaccination; however, a safe, efficacious and cost-effective vaccine against C. trachomatis infection remains an unmet medical need. C. trachomatis major outer membrane protein (MOMP), a ß-barrel integral outer membrane protein, is the most abundant antigen in the outer membrane of the bacterium and has been evaluated as a subunit vaccine candidate. Recombinant MOMP (rMOMP) expressed in E. coli cytoplasm forms inclusion bodies and rMOMP extracted from inclusion bodies results in a reduced level of protection compared to the native MOMP in a mouse challenge model. RESULTS: We sought to target the recombinant expression of MOMP to the E. coli outer membrane (OM). Successful surface expression was achieved with codon harmonization, utilization of low copy number vectors and promoters with moderate strength, suitable leader sequences and optimization of cell culture conditions. rMOMP was extracted from E. coli outer membrane, purified, and characterized biophysically. The OM expressed and purified rMOMP is immunogenic in mice and elicits antibodies that react to the native antigen, Chlamydia elementary body (EB). CONCLUSIONS: C. trachomatis MOMP was functionally expressed on the surface of E. coli outer membrane. The OM expressed and purified rMOMP elicits antibodies that react to the native antigen, Chlamydia EB, in a mouse immunogenicity model. Surface expression of MOMP could provide useful reagents for vaccine research, and the methodology could serve as a platform to produce other outer membrane proteins recombinantly.

Discovery and Characterization of Phage Display-Derived Human Monoclonal Antibodies against RSV F Glycoprotein.

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in infants, the elderly and in immunosuppressed populations. The vast majority of neutralizing antibodies isolated from human subjects target the RSV fusion (F) glycoprotein, making it an attractive target for the development of vaccines and therapeutic antibodies. Currently, Synagis® (palivizumab) is the only FDA approved antibody drug for the prevention of RSV infection, and there is a great need for more effective vaccines and therapeutics. Phage display is a powerful tool in antibody discovery with the advantage that it does not require samples from immunized subjects. In this study, Morphosys HuCAL GOLD® phage libraries were used for panning against RSV prefusion and postfusion F proteins. Panels of human monoclonal antibodies (mAbs) against RSV F protein were discovered following phage library panning and characterized. Antibodies binding specifically to prefusion or postfusion F proteins and those binding both conformations were identified. 3B1 is a prototypic postfusion F specific antibody while 2E1 is a prototypic prefusion F specific antibody. 2E1 is a potent broadly neutralizing antibody against both RSV A and B strains. Epitope mapping experiments identified a conformational epitope spanning across three discontinuous sections of the RSV F protein, as well as critical residues for antibody interaction.

Protection of rhesus macaques against inhalational anthrax with a Bacillus anthracis capsule conjugate vaccine.

The efficacy of currently licensed anthrax vaccines is largely attributable to a single Bacillus anthracis immunogen, protective antigen. To broaden protection against possible strains resistant to protective antigen-based vaccines, we previously developed a vaccine in which the anthrax polyglutamic acid capsule was covalently conjugated to the outer membrane protein complex of Neisseria meningitidis serotype B and demonstrated that two doses of 2.5µg of this vaccine conferred partial protection of rhesus macaques against inhalational anthrax . Here, we demonstrate complete protection of rhesus macaques against inhalational anthrax with a higher 50µg dose of the same capsule conjugate vaccine. These results indicate that B. anthracis capsule is a highly effective vaccine component that should be considered for incorporation in future generation anthrax vaccines.

Design and Purification of Subunit Vaccines for Prevention of Clostridium difficile Infection.

Clostridium difficile is a gram-positive bacterium responsible for a large proportion of nosocomial infections in the developed world. C. difficile secretes toxins A and B (TcdA and TcdB) and both toxins act synergistically to induce a spectrum of pathological responses in infected individuals ranging from pseudomembranous colitis to C. difficile-associated diarrhea. Toxins A and B have been actively investigated as components of prophylactic vaccine as well as targets for therapeutic intervention with antibodies. Expression of such toxins by recombinant technology is often difficult and may require special handling and adherence to strict safety regulations during the manufacturing process due to the inherent toxicity of the proteins. Both toxins are large proteins (308 kDa and 270 kDa, respectively) and contain distinct domains mediating cell attachment, cellular translocation, and enzymatic (glucosidase) activity. Here we describe methods to produce fragments of Toxin B for their subsequent evaluation as components of experimental C. difficile vaccines. Methods presented include selection of fragments encompassing distinct functional regions of Toxin B, purification methods to yield high quality proteins, and analytical evaluation techniques. The approach presented focuses on Toxin B but could be applied to the other component, Toxin A, and/or to any difficult to express or toxic protein.

Preclinical evaluations of peptide-conjugate vaccines targeting the antigenic domain-2 of glycoprotein B of human cytomegalovirus.

The Antigenic Domain 2 (AD-2) is a short region near the N-terminus of glycoprotein B of human cytomegalovirus (HCMV). AD-2 has been shown to contain linear epitopes that are targets for neutralizing monoclonal antibodies from human subjects with natural HCMV infection. However, AD-2 appears to be masked by the adjacent immunodominant AD-1 region. We assessed a serum panel from HCMV-seropositive individuals and found a wide range of antibody titers to AD-2; these did not correlate to serum neutralization. To expose potential epitopes in AD-2, we constructed a series of AD-2 peptide-conjugate vaccines. Mice were immunized 3 times and produced high and sustained antibody titers to AD-2 peptides, but neutralization was weak even after a single boost with whole HCMV virions. Rabbits were likewise immunized with AD-2 peptide vaccines, and produced a robust antibody response, but neutralization was inferior to a recombinant gB vaccine with an oil-in-water adjuvant. These results highlight the challenges of developing a peptide-based vaccine specific to the HCMV gB AD-2 region.

Development of a recombinant toxin fragment vaccine for Clostridium difficile infection.

Clostridium difficile infection (CDI) is the major cause of antibiotic-associated diarrhea and pseudomembranous colitis, a disease associated with significant morbidity and mortality. The disease is mostly of nosocomial origin, with elderly patients undergoing anti-microbial therapy being particularly at risk. C. difficile produces two large toxins: Toxin A (TcdA) and Toxin B (TcdB). The two toxins act synergistically to damage and impair the colonic epithelium, and are primarily responsible for the pathogenesis associated with CDI. The feasibility of toxin-based vaccination against C. difficile is being vigorously investigated. A vaccine based on formaldehyde-inactivated Toxin A and Toxin B (toxoids) was reported to be safe and immunogenic in healthy volunteers and is now undergoing evaluation in clinical efficacy trials. In order to eliminate cytotoxic effects, a chemical inactivation step must be included in the manufacturing process of this toxin-based vaccine. In addition, the large-scale production of highly toxic antigens could be a challenging and costly process. Vaccines based on non-toxic fragments of genetically engineered versions of the toxins alleviate most of these limitations. We have evaluated a vaccine assembled from two recombinant fragments of TcdB and explored their potential as components of a novel experimental vaccine against CDI. Golden Syrian hamsters vaccinated with recombinant fragments of TcdB combined with full length TcdA (Toxoid A) developed high titer IgG responses and potent neutralizing antibody titers. We also show here that the recombinant vaccine protected animals against lethal challenge with C. difficile spores, with efficacy equivalent to the toxoid vaccine. The development of a two-segment recombinant vaccine could provide several advantages over toxoid TcdA/TcdB such as improvements in manufacturability.

Sporozoite neutralizing antibodies elicited in mice and rhesus macaques immunized with a Plasmodium falciparum repeat peptide conjugated to meningococcal outer membrane protein complex.

Antibodies that neutralize infectivity of malaria sporozoites target the central repeat region of the circumsporozoite (CS) protein, which in Plasmodium falciparum is comprised primarily of 30-40 tandem NANP tetramer repeats. We evaluated immunogenicity of an alum-adsorbed (NANP)(6) peptide conjugated to an outer membrane protein complex (OMPC) derived from Neisseria meningitidis, a carrier protein used in a licensed Haemophilus influenzae pediatric vaccine. Mice immunized with (NANP)(6)-OMPC adsorbed to Merck's alum adjuvant (MAA), with or without Iscomatrix® as co-adjuvant, developed high levels of anti-repeat peptide antibody that inhibited in vitro invasion of human hepatoma cells by transgenic P. berghei sporozoites that express P. falciparum CS repeats (PfPb). Inhibition of sporozoite invasion in vitro correlated with in vivo resistance to challenge by the bites of PfPb-infected mosquitoes. Challenged mice had >90% reduction of hepatic stage parasites as measured by real-time PCR, and either sterile immunity, i.e., no detectable blood stage parasites, or delayed prepatent periods which indicate neutralization of a majority, but not all, sporozoites. Rhesus macaques immunized with two doses of (NANP)(6)-OMPC/MAA formulated with Iscomatrix® developed anti-repeat antibodies that persisted for ~2 years. A third dose of (NANP)(6)-OMPC/MAA+ Iscomatrix® at that time elicited strong anamnestic antibody responses. Rhesus macaque immune sera obtained post second and third dose of vaccine displayed high levels of sporozoite neutralizing activity in vitro that correlated with presence of high anti-repeat antibody titers. These preclinical studies in mice of different MHC haplotypes and a non-human primate support use of CS peptide-OMPC conjugates as a highly immunogenic platform to evaluate CS protective epitopes. Potential pre-erythrocytic vaccines can be combined with sexual blood stage vaccines as a multi-antigen malaria vaccine to block invasion and transmission of Plasmodium parasites.

Identification and characterization of pleckstrin-homology-domain-dependent and isoenzyme-specific Akt inhibitors.

We developed a high-throughput HTRF (homogeneous time-resolved fluorescence) assay for Akt kinase activity and screened approx. 270000 compounds for their ability to inhibit the three isoforms of Akt. Two Akt inhibitors were identified that exhibited isoenzyme specificity. The first compound (Akt-I-1) inhibited only Akt1 (IC50 4.6 microM) while the second compound (Akt-I-1,2) inhibited both Akt1 and Akt2 with IC50 values of 2.7 and 21 microM respectively. Neither compound inhibited Akt3 nor mutants lacking the PH (pleckstrin homology) domain at concentrations up to 250 microM. These compounds were reversible inhibitors, and exhibited a linear mixed-type inhibition against ATP and peptide substrate. In addition to inhibiting kinase activity of individual Akt isoforms, both inhibitors blocked the phosphorylation and activation of the corresponding Akt isoforms by PDK1 (phosphoinositide-dependent kinase 1). A model is proposed in which these inhibitors bind to a site formed only in the presence of the PH domain. Binding of the inhibitor is postulated to promote the formation of an inactive conformation. In support of this model, antibodies to the Akt PH domain or hinge region blocked the inhibition of Akt by Akt-I-1 and Akt-I-1,2. These inhibitors were found to be cell-active and to block phosphorylation of Akt at Thr308 and Ser473, reduce the levels of active Akt in cells, block the phosphorylation of known Akt substrates and promote TRAIL (tumour-necrosis-factor-related apoptosis-inducing ligand)-induced apoptosis in LNCap prostate cancer cells.