Report of the Assay Guidance Workshop on 3-Dimensional Tissue Models for Antiviral Drug Development.

The National Center for Advancing Translational Sciences (NCATS) Assay Guidance Manual (AGM) Workshop on 3D Tissue Models for Antiviral Drug Development, held virtually on 7-8 June 2022, provided comprehensive coverage of critical concepts intended to help scientists establish robust, reproducible, and scalable 3D tissue models to study viruses with pandemic potential. This workshop was organized by NCATS, the National Institute of Allergy and Infectious Diseases, and the Bill and Melinda Gates Foundation. During the workshop, scientific experts from academia, industry, and government provided an overview of 3D tissue models' utility and limitations, use of existing 3D tissue models for antiviral drug development, practical advice, best practices, and case studies about the application of available 3D tissue models to infectious disease modeling. This report includes a summary of each workshop session as well as a discussion of perspectives and challenges related to the use of 3D tissues in antiviral drug discovery.

Immune Prophylaxis and Therapy for Human Cytomegalovirus Infection.

Human Cytomegalovirus (HCMV) infection is widespread and can result in severe sequelae in susceptible populations. Primary HCMV infection of naïve individuals results in life-long latency characterized by frequent and sporadic reactivations. HCMV infection elicits a robust antibody response, including neutralizing antibodies that can block the infection of susceptible cells in vitro and in vivo. Thus, antibody products and vaccines hold great promise for the prevention and treatment of HCMV, but to date, most attempts to demonstrate their safety and efficacy in clinical trials have been unsuccessful. In this review we summarize publicly available data on these products and highlight new developments and approaches that could assist in successful translation of HCMV immunotherapies.

Combined thrombogenic effects of vessel injury, pregnancy and procoagulant immune globulin administration in mice.

BACKGROUND: Pregnant women are at increased risk of thrombotic adverse events. Plasma derived immune globulin (IG) products, which are used in pregnancy for various indications, may contain procoagulant impurity activated coagulation factor XI (FXIa). Procoagulant IG products have been associated with increased thrombogenicity but their effect in pregnancy is unknown. METHODS: Late pregnant (gestation days 17-20) or early lactation (days 1-3) and control female mice were treated with IGs supplemented with human FXIa then subjected to ferric chloride (FeCl3) vessel injury. Occlusion of blood vessel was assessed by recording blood velocity in the femoral vein for 20 min using doppler ultrasound laser imaging. FXIa dose was selected by the ability to increase thrombin generation in mouse plasma in vitro. RESULTS: FXIa produced robust thrombin generation in mouse plasma ex vivo. Following FeCl3 injury, pregnant and non-pregnant mice receiving IG + FXIa exhibited faster reduction of blood velocity in femoral vein compared to IG alone or untreated controls. In vitro, thrombin generation in plasma samples collected after thrombosis in FXIa-treated animals was elevated and could be reduced by anti-FXI antibody. CONCLUSIONS: Our results suggest that intravenously-administered FXIa may contribute to thrombosis at the site of vascular injury in both pregnant and non-pregnant animals.

Binding and neutralizing anti-cytomegalovirus activities in immune globulin products.

Congenital infection as well as infection of immunocompromised individuals by cytomegalovirus (CMV) can be associated with significant morbidity, mortality, and long-term adverse health outcomes. Assessment of anti-viral activity using appropriate assays is essential for ensuring safe and efficacious use of therapeutic CMV immune globulin (IG) products. In this study, we used commercial ELISA kits to compare anti-CMV antibody binding activity and avidity for lots of CMV-specific and normal IG products available in the US market. Additionally, neutralizing activity of IG products was measured against CMV strains (AD169wt131 or TB40E-GFP) in MRC-5 human fibroblasts and ARPE-19 human epithelial cells. Our data revealed that, regardless of the method, anti-CMV activity was higher in CMV IG lots we tested compared with normal IG lots; CMV binding activity was at least 4-fold higher, and neutralizing activity at least 2- and 3-fold higher for epithelial and fibroblast cells, respectively, in CMV IG lots compared with normal IG lots. Furthermore, anti-CMV activity values from all three methods (ELISA, neutralization in MRC-5 cells, and neutralization in ARPE-19 cells) were highly correlated, whereas avidity, although higher in CMV IG lots, did not correlate well with either binding or neutralizing activities.

Mapping the binding region on the low density lipoprotein receptor for blood coagulation factor VIII.

Low density lipoprotein receptor (LDLR) was shown to mediate clearance of blood coagulation factor VIII (FVIII) from the circulation. To elucidate the mechanism of interaction of LDLR and FVIII, our objective was to identify the region of the receptor necessary for binding FVIII. Using surface plasmon resonance, we found that LDLR exodomain and its cluster of complement-type repeats (CRs) bind FVIII in the same mode. This indicated that the LDLR site for FVIII is located within the LDLR cluster. Similar results were obtained for another ligand of LDLR, α-2-macroglobulin receptor-associated protein (RAP), a common ligand of receptors from the LDLR family. We further generated a set of recombinant fragments of the LDLR cluster and assessed their structural integrity by binding to RAP and by circular dichroism. A number of fragments overlapping CR.2-5 of the cluster were positive for binding RAP and FVIII. The specificity of these interactions was tested by site-directed mutagenesis of conserved tryptophans within the LDLR fragments. For FVIII, the specificity was also tested using a single-chain variable antibody fragment directed against the FVIII light chain as a competitor. Both cases resulted in decreased binding, thus confirming its specificity. The mutagenic study also showed an importance of the conserved tryptophans in LDLR for both ligands, and the competitive binding results showed an involvement of the light chain of FVIII in its interaction with LDLR. In conclusion, the region of CR.2-5 of LDLR was defined as the binding site for FVIII and RAP.

Pharmacokinetics of antibodies during Pregnancy: Impact of pregnancy on the pharmacokinetics of antibodies (Part 2).

In Part 1, we provided a general description of macromolecules, pharmacokinetics (PK) characteristics in non-pregnant subjects, and the physiological changes during pregnancy. Here we further elaborate on the impact of pregnancy on the PK of antibodies through illustrative case studies (immunoglobulins, infliximab, adalimumab and eculizumab). Using published data from nonclinical and clinical studies, we present measured or calculated PK parameters from pregnant subjects comparing with data from non-pregnant subjects, if available. Due to the paucity of PK data evaluating PK of antibodies during pregnancy, we also provide examples of PK studies for small molecules. Finally, we draw conclusions on the nature and direction of PK changes for both antibodies and small molecules as well as provide recommendations for areas that would benefit from further studies.

Pharmacokinetics of antibodies during pregnancy: General pharmacokinetics and pregnancy related physiological changes (Part 1).

Pharmacokinetics (PK) studies are important to determine a safe and effective dose of both small and large molecule drugs. Intrinsic factors such as pregnancy can substantially alter the PK of a drug. Several PK studies have been published for small molecules administered during pregnancy, but such investigations are scarce for macromolecules including monoclonal and polyclonal antibodies. In this part 1 of 2 reviews, we first provide a general description of macromolecule drugs, the PK differences with small molecules, and current knowledge on their absorption, distribution, metabolism and elimination in non-pregnant subjects. We then review in detail the physiological changes during pregnancy. While some of the physiologic adaptions of pregnancy, for example increased plasma volume and cardiac output, are expected to impact PK of antibody therapeutics, the effects of others, such as increased GFR and altered immune responses are not fully understood. We conclude that further investigations are needed to fully elucidate how pregnancy can impact PK properties of macromolecules.

Uses and Challenges of Antiviral Polyclonal and Monoclonal Antibody Therapies.

Viral diseases represent a major public health concerns and ever-present risks for developing into future pandemics. Antiviral antibody therapeutics, either alone or in combination with other therapies, emerged as valuable preventative and treatment options, including during global emergencies. Here we will discuss polyclonal and monoclonal antiviral antibody therapies, focusing on the unique biochemical and physiological properties that make them well-suited as therapeutic agents. We will describe the methods of antibody characterization and potency assessment throughout development, highlighting similarities and differences between polyclonal and monoclonal products as appropriate. In addition, we will consider the benefits and challenges of antiviral antibodies when used in combination with other antibodies or other types of antiviral therapeutics. Lastly, we will discuss novel approaches to the characterization and development of antiviral antibodies and identify areas that would benefit from additional research.

Nanoluciferase Reporter Zika Viruses as Tools for Assessing Infection Kinetics and Antibody Potency.

Zika virus (ZIKV) has become endemic in multiple tropical and subtropical regions and has the potential to become widespread in countries with limited prior exposure to this infection. One of the most concerning sequelae of ZIKV infection is the teratogenic effect on the developing fetus, with the mechanisms of viral spread to and across the placenta remaining largely unknown. Although vaccine trials and prophylactic or therapeutic treatments are being studied, there are no approved treatments or vaccines for ZIKV. Appropriate tests, including potency and in vivo assays to assess the safety and efficacy of these modalities, can greatly aid both the research of the pathophysiology of the infection and the development of anti-ZIKV therapeutics. Building on previous work, we tested reporter ZIKV variants that express nanoluciferase in cell culture and in vivo assays. We found that these variants can propagate in cells shown to be susceptible to the widely used clinical isolate PRVABC59, including Vero and human placenta cell lines. When used in neutralization assays with bioluminescence as readout, these variants gave rise to neutralization curves similar to those produced by PRVABC59, while being better suited for performing high-throughput assays. In addition, the engineered reporter variants can be useful research tools when used in other in vitro and in vivo assays, as we illustrated in transcytosis experiments and a pilot study in guinea pigs.

Epitope mapping by random peptide phage display reveals essential residues for vaccinia extracellular enveloped virion spread.

BACKGROUND: A33 is a type II integral membrane protein expressed on the extracellular enveloped form of vaccinia virus (VACV). Passive transfer of A33-directed monoclonal antibodies or vaccination with an A33 subunit vaccine confers protection against lethal poxvirus challenge in animal models. Homologs of A33 are highly conserved among members of the Orthopoxvirus genus and are potential candidates for inclusion in vaccines or assays targeting extracellular enveloped virus activity. One monoclonal antibody directed against VACV A33, MAb-1G10, has been shown to target a conformation-dependent epitope. Interestingly, while it recognizes VACV A33 as well as the corresponding variola homolog, it does not bind to the monkeypox homolog. In this study, we utilized a random phage display library to investigate the epitope recognized by MAb-1G10 that is critical for facilitating cell-to-cell spread of the vaccinia virus. RESULTS: By screening with linear or conformational random phage libraries, we found that phages binding to MAb-1G10 display the consensus motif CEPLC, with a disulfide bond formed between two cysteine residues required for MAb-1G10 binding. Although the phage motif contained no linear sequences homologous to VACV A33, structure modeling and analysis suggested that residue D115 is important to form the minimal epitope core. A panel of point mutants expressing the ectodomain of A33 protein was generated and analyzed by either binding assays such as ELISA and immunoprecipitation or a functional assessment by blocking MAb-1G10 mediated comet inhibition in cell culture. CONCLUSIONS: These results confirm L118 as a component of the MAb-1G10 binding epitope, and further identify D115 as an essential residue. By defining the minimum conformational structure, as well as the conformational arrangement of a short peptide sequence recognized by MAb-1G10, these results introduce the possibility of designing small molecule mimetics that may interfere with the function of A33 in vivo. This information will also be useful for designing improved assays to evaluate the potency of monoclonal and polyclonal products that target A33 or A33-modulated EV dissemination.

Passive Immunoprophylaxis for the Protection of the Mother and Her Baby: Insights from In Vivo Models of Antibody Transport.

Pregnant women are at high risk for infection by pathogens. Vertical transmission of infectious agents, such as Zika, hepatitis B, and cytomegalovirus during pregnancy, remains a public health problem, associated with dire outcomes for the neonate. Thus, a safe prophylactic and therapeutic approach for protecting the mother and the neonate from infections remains a high priority. Our work is focused on better understanding the safety and efficacy determinants of IgG antibody preparations when used during pregnancy to benefit the mother and her baby. Using pregnant guinea pigs, we demonstrated that biodistribution of administered IgG to the fetus increases with gestation and results in lower maternal and higher fetal antibody concentrations as pregnancy progresses. Data suggests that partition of antibody immunotherapy to the fetal compartment may contribute to a lower maternal exposure (as measured by the AUC) and a shorter mean residence time of the IgG therapeutic at the end of pregnancy compared to nonpregnant age-matched controls, irrespective of the administered dose. Our studies provide insights on the importance of selecting an efficacious dose in pregnancy that takes into account IgG biodistribution to the fetus. The use of appropriate animal models of placental transfer and infectious disease during pregnancy would facilitate pharmacokinetic modeling to derive a starting dose in clinical trials.

Gestation age dependent transfer of human immunoglobulins across placenta in timed-pregnant guinea pigs.

INTRODUCTION: When administered during pregnancy, antibodies and other biologic drugs that contain the Fc part of the IgG molecule can traverse the placenta. Although it is generally accepted that the FcRn receptor mediates this process, gaps remain in our understanding of underlying details in humans and in common laboratory animal species. METHODS: We expanded our previous studies in timed-pregnant guinea pigs to both measure the transport of human (h) IgG at earlier gestation ages in vivo and evaluate FcRn function in vitro using Surface Plasmon Resonance (SPR) and Madin-Darby canine kidney cells (MDCK) that express guinea pig (gp) FcRn. RESULTS: In timed-pregnant guinea pigs both the average concentration of hIgG in the fetus and its ratio to maternal hIgG concentration increase exponentially with gestation age. Thus, hIgG fetal:maternal concentration ratios increase from an average of 1% to 3%, 17%, and 76% on GD ∼26, 35, 46, and 54, respectively. In vitro, gpFcRn immobilized on a solid surface can bind hIgG and gpIgG preparations in a similar manner. All engineered human Fc isotype-specific constructs were internalized by MDCK-gpFcRn cells at significant levels. While not significant, their recycling and hIgG transcytosis by this cell line also trend higher than background controls. DISCUSSION: Pregnant guinea pigs exhibit similarities with humans in the degree and timing of transplacental transfer as well as the ability of their FcRn to bind and internalize hIgG in vitro. Further studies are needed to guide building appropriate systems for the evaluation of FcRn mediated function of human immunoglobulin therapies.

Transplacental Transfer of Hepatitis B Neutralizing Antibody during Pregnancy in an Animal Model: Implications for Newborn and Maternal Health.

Despite the success of postexposure prophylaxis (PEP) of the newborn in preventing mother-to-child transmission of hepatitis B virus), in non-US clinical trials, administering hepatitis B immune globulin (HBIG) to mothers at the end of pregnancy (in addition to passive-active PEP of the newborn) only partially improved outcomes. That is, a significant percentage of newborns became infected during their first year of life. We used a relevant animal model for human IgG transplacental transfer to study dose, time and subclass dependence of HBV neutralizing antibody (nAb) maternal, and fetal levels at the end of pregnancy. Pregnant guinea pigs received 50 or 100 IU/kg HBIGIV 2-5 days before delivery. Human total IgG, IgG subclasses, and nAb in mothers and their litters were measured. In vitro analyses of guinea pig Fc neonatal receptor binding to HBIGIV, as well as to all human IgG subclasses, were also performed. Our study showed that nAb transferred transplacentally from the pregnant guinea pigs to their litters; no transfer occurred during parturition. The amount of the transferred nAb was dose and time dependent. Thus, selection of an efficacious dose in the clinic is important: microdosing may be underdosing, particularly in cases of high viraemia.