Inhaled "Muco-Trapping" Monoclonal Antibody Effectively Treats Established Respiratory Syncytial Virus (RSV) Infections.

Respiratory syncytial virus (RSV) causes substantial morbidity and mortality in infants, the immunocompromised, and the elderly. RSV infects the airway epithelium via the apical membrane and almost exclusively sheds progeny virions back into the airway mucus (AM), making RSV difficult to target by systemically administered therapies. An inhalable "muco-trapping" variant of motavizumab (Mota-MT), a potent neutralizing mAb against RSV F is engineered. Mota-MT traps RSV in AM via polyvalent Fc-mucin bonds, reducing the fraction of fast-moving RSV particles in both fresh pediatric and adult AM by ≈20-30-fold in a Fc-glycan dependent manner, and facilitates clearance from the airways of mice within minutes. Intranasal dosing of Mota-MT eliminated viral load in cotton rats within 2 days. Daily nebulized delivery of Mota-MT to RSV-infected neonatal lambs, beginning 3 days after infection when viral load is at its maximum, led to a 10 000-fold and 100 000-fold reduction in viral load in bronchoalveolar lavage and lung tissues relative to placebo control, respectively. Mota-MT-treated lambs exhibited reduced bronchiolitis, neutrophil infiltration, and airway remodeling than lambs receiving placebo or intramuscular palivizumab. The findings underscore inhaled delivery of muco-trapping mAbs as a promising strategy for the treatment of RSV and other acute respiratory infections.

ZB-06, a vaginal film containing an engineered human contraceptive antibody (HC4-N), demonstrates safety and efficacy in a phase 1 postcoital test and safety study.

BACKGROUND: With an unplanned pregnancy rate of 50% or more in many countries, there is an urgent need for contraceptives that are more accessible and acceptable. To meet the growing demand for new contraceptives, ZabBio developed ZB-06, a vaginal film containing HC4-N, a human contraceptive antibody that inactivates sperm. OBJECTIVE: This study aimed to assess the potential contraceptive activity of the ZB-06 film using a surrogate assessment for contraceptive efficacy, the postcoital test. We also assessed clinical safety of film use among healthy heterosexual couples. Serum, cervical mucus, and vaginal fluid HC4-N antibody concentrations and sperm agglutination potency were determined after single film use. Changes in the concentration of soluble proinflammatory cytokines and vaginal Nugent score after film use were measured as subclinical safety endpoints. STUDY DESIGN: This was a phase 1, first-in-woman, open-label, proof-of-concept, postcoital test and safety study. RESULTS: A total of 20 healthy women were enrolled in the study, and 8 heterosexual couples completed all study visits. The product was safe for both female participants and their male sexual partners. The postcoital test performed on ovulatory cervical mucus at baseline (no product use) revealed a mean of 25.9 (±30.6) progressively motile sperm per high-power field. After use of a single ZB-06 film before intercourse, this number dropped to 0.04 (±0.06) progressively motile sperm per high-power field (P<.0001). At the follow-up postcoital test visit approximately 1 month later (no product use), a mean of 47.4 (±37.4) progressively motile sperm per high-power field was observed, indicating contraceptive reversibility. CONCLUSION: A single dose of the ZB-06 film applied before intercourse was safe and met efficacy surrogate benchmarks of excluding progressively motile sperm from ovulatory cervical mucus. These data indicate that ZB-06 is a viable contraceptive candidate warranting further development and testing.

LALAPG variant of the Human Contraception Antibody (HCA) reduces Fc-mediated effector functions while maintaining sperm agglutination activity.

High rates of unintended pregnancies worldwide indicate a need for more accessible and acceptable methods of contraception. We have developed a monoclonal antibody, the Human Contraception Antibody (HCA), for use by women in vaginal films and rings for contraception. The divalent F(ab')2 region of HCA binds to an abundant male reproductive tract-specific antigen, CD52g, and potently agglutinates sperm. Certain other antibody activities mediated by the Fc region such as mucus trapping, complement-dependent cytotoxicity (CDC) and antibody-dependent cellular phagocytosis (ADCP) could have beneficial or negative effects. The purpose of this study was to document HCA Fc effector functions and determine whether an engineered variant of HCA with a modified Fc region, HCA-LALAPG, retains desirable contraceptive activity while minimizing Fc-mediated effects. Fab and Fc functions were compared between HCA and HCA-LALAPG. Fab activity was assessed using sperm agglutination and modified swim-up ("sperm escape") assays. Fc functions were assessed by CDC (sperm immobilization), ADCP, and cervical mucus penetration assays. HCA and HCA-LALAPG showed equivalent activity in assays of Fab function. In the assays of Fc function, HCA supported strong CDC, ADCP, and sperm trapping in cervical mucus whereas HCA-LALAPG demonstrated little to no activity. HCA and the HCA-LALAPG variant were both highly effective in the sperm agglutination assays but differed in Fc mediated functions. Use of the HCA-LALAPG variant for contraception in women could reduce antibody-mediated inflammation and antigen presentation but may have reduced contraceptive efficacy due to much weaker sperm trapping in mucus and complement-dependent sperm immobilization activity.

Hexavalent sperm-binding IgG antibody released from vaginal film for development of potent on-demand nonhormonal female contraception.

Nonhormonal products for on-demand contraception are a global health technology gap; this unmet need motivated us to pursue the use of sperm-binding monoclonal antibodies to enable effective on-demand contraception. Here, using the cGMP-compliant Nicotiana-expression system, we produced an ultrapotent sperm-binding IgG antibody possessing 6 Fab arms per molecule that bind a well-established contraceptive antigen target, CD52g. We term this hexavalent antibody "Fab-IgG-Fab" (FIF). The Nicotiana-produced FIF had at least 10-fold greater sperm-agglutination potency and kinetics than the parent IgG, while preserving Fc-mediated trapping of individual spermatozoa in mucus. We formulated the Nicotiana-produced FIF into a polyvinyl alcohol-based water-soluble contraceptive film and evaluated its potency in reducing progressively motile sperm in the sheep vagina. Two minutes after vaginal instillation of human semen, no progressively motile sperm were recovered from the vaginas of sheep receiving FIF Film. Our work supports the potential of multivalent contraceptive antibodies to provide safe, effective, on-demand nonhormonal contraception.

Engineering sperm-binding IgG antibodies for the development of an effective nonhormonal female contraception.

Many women risk unintended pregnancy because of medical contraindications or dissatisfaction with contraceptive methods, including real and perceived side effects associated with the use of exogenous hormones. We pursued direct vaginal delivery of sperm-binding monoclonal antibodies (mAbs) that can limit progressive sperm motility in the female reproductive tract as a strategy for effective nonhormonal contraception. Here, motivated by the greater agglutination potencies of polyvalent immunoglobulins but the bioprocessing ease and stability of immunoglobulin G (IgG), we engineered a panel of sperm-binding IgGs with 6 to 10 antigen-binding fragments (Fabs), isolated from a healthy immune-infertile woman against a unique surface antigen universally present on human sperm. These highly multivalent IgGs (HM-IgGs) were at least 10- to 16-fold more potent and faster at agglutinating sperm than the parent IgG while preserving the crystallizable fragment (Fc) of IgG that mediates trapping of individual spermatozoa in mucus. The increased potencies translated into effective (>99.9%) reduction of progressively motile sperm in the sheep vagina using as little as 33 µg of the 10-Fab HM-IgG. HM-IgGs were produced at comparable yields and had identical thermal stability to the parent IgG, with greater homogeneity. HM-IgGs represent not only promising biologics for nonhormonal contraception but also a promising platform for engineering potent multivalent mAbs for other biomedical applications.

Production and characterization of a human antisperm monoclonal antibody against CD52g for topical contraception in women.

BACKGROUND: Approximately 40% of human pregnancies are unintended, indicating a need for more acceptable effective contraception methods. New antibody production systems make it possible to manufacture reagent-grade human monoclonal antibodies (mAbs) for clinical use. We used the Nicotiana platform to produce a human antisperm mAb and tested its efficacy for on-demand topical contraception. METHODS: Heavy and light chain variable region DNA sequences of a human IgM antisperm antibody derived from an infertile woman were inserted with human IgG1 constant region sequences into an agrobacterium and transfected into Nicotiana benthamiana. The product, an IgG1 mAb ["Human Contraception Antibody" (HCA)], was purified on Protein A columns, and QC was performed using the LabChip GXII Touch protein characterization system and SEC-HPLC. HCA was tested for antigen specificity by immunofluorescence and western blot assays, antisperm activity by sperm agglutination and complement dependent sperm immobilization assays, and safety in a human vaginal tissue (EpiVaginal™) model. FINDINGS: HCA was obtained at concentrations ranging from 0.4 to 4 mg/ml and consisted of > 90% IgG monomers. The mAb specifically reacted with a glycan epitope on CD52g, a glycoprotein produced in the male reproductive tract and found in abundance on sperm. HCA potently agglutinated sperm under a variety of relevant physiological conditions at concentrations ≥ 6.25 µg/ml, and mediated complement-dependent sperm immobilization at concentrations ≥ 1 µg/ml. HCA and its immune complexes did not induce inflammation in EpiVaginal™ tissue. INTERPRETATION: HCA, an IgG1 mAb with potent sperm agglutination and immobilization activity and a good safety profile, is a promising candidate for female contraception. FUNDING: This research was supported by grants R01 HD095630 and P50HD096957 from the National Institutes of Health.

Safety, acceptability, and pharmacokinetics of a monoclonal antibody-based vaginal multipurpose prevention film (MB66): A Phase I randomized trial.

BACKGROUND: MB66 film is a multipurpose prevention technology (MPT) product with monoclonal antibodies (mAbs) against HIV-1 (VRC01-N) and HSV-1 and 2 (HSV8-N). The mAbs were produced by transient expression in Nicotiana benthamiana (N). We conducted a Phase I clinical trial to assess the safety, pharmacokinetics (PK), and ex vivo efficacy of single and repeated doses of MB66 when used intravaginally. METHODS AND FINDINGS: The clinical trial enrolled healthy reproductive-aged, sexually abstinent women. In Segment A, 9 women received a single MB66 film which was inserted into the vaginal posterior fornix by a clinician. In Segment B, 29 women were randomly assigned to MB66 (Active) or Placebo film groups and were instructed to insert 1 film vaginally for 7 consecutive days. Visits and clinical sampling occurred predose and at various time points after single and repeated film doses. The primary endpoint was number of adverse events (AEs) Grade 2 or higher related to product use. Secondary endpoints included film dissolution rate, Nugent score (a Gram stain scoring system to diagnose bacterial vaginosis), vaginal pH, post-use survey results, cytokine concentrations in cervicovaginal lavage (CVL) specimens (assessed by Luminex assay), mAb concentrations in vaginal fluid collected from 4 sites (assessed by ELISA), and HIV and HSV neutralization activity of CVL samples ex vivo (assessed by TZM-bl and plaque reduction assay, respectively). The product was generally safe and well tolerated, with no serious AEs recorded in either segment. The AEs in this study were primarily genitourinary in nature with the most commonly reported AE being asymptomatic microscopic hematuria. There were no differences in vaginal pH or Nugent scores or significant increases in levels of proinflammatory cytokines for up to 7 days after film insertion in either segment or between Active and Placebo groups. Acceptability and willingness to use the product were judged to be high by post-use surveys. Concentrations of VRC01-N and HSV8-N in vaginal secretions were assessed over time to generate pharmacokinetic curves. Antibody levels peaked 1 hour postdosing with Active film (median: 35 µg/mL) and remained significantly elevated at 24 hours post first and seventh film (median: 1.8 µg/mL). Correcting for sample dilution (1:20), VRC01-N concentrations ranged from 36 to 700 µg/mL at the 24-hour time point, greater than 100-fold the IC50 for VRC01 (0.32 µg/mL); HSV8-N concentrations ranged from 80 to 601 µg/mL, well above the IC50 of 0.1 µg/m. CVL samples collected 24 hours after MB66 insertion significantly neutralized both HIV-1 and HSV-2 ex vivo. Study limitations include the small size of the study cohort, and the fact that no samples were collected between 24 hours and 7 days for pharmacokinetic evaluation. CONCLUSIONS: Single and repeated intravaginal applications of MB66 film were safe, well tolerated, and acceptable. Concentrations and ex vivo bioactivity of both mAbs in vaginal secretions were significantly elevated and thus could provide protection for at least 24 hours postdose. However, further research is needed to evaluate the efficacy of MB66 film in women at risk for HIV and HSV infection. Additional antibodies could be added to this platform to provide protection against other sexually transmitted infections (STIs) and contraception. TRIAL REGISTRATION: ClinicalTrials.gov NCT02579083.

Engineering tetravalent IgGs with enhanced agglutination potencies for trapping vigorously motile sperm in mucin matrix.

Multivalent antibodies such as sIgA can crosslink motile entities such as sperm and bacteria, creating agglomerates that are too large to permeate the dense mucin matrix in mucus, a process commonly referred to as immune exclusion. Unfortunately, sIgA remains challenging to produce in large quantities, and easily aggregates, which prevented their use in clinical applications. To develop sIgA-like tetravalent antibodies that are stable and can be easily produced in large quantities, we designed two IgGs possessing 4 identical Fab domains, with the Fabs arranged either in serial or in the diametrically opposite orientation. As a proof-of-concept, we engineered these tetravalent IgG constructs to bind a ubiquitous sperm antigen using a Fab previously isolated from an immune infertile woman. Both constructs possess at least 4-fold greater agglutination potency and induced much more rapid sperm agglutination than the parent IgG, while exhibiting comparable production yields and identical thermostability as the parent IgG. These tetravalent IgGs offer promise for non-hormonal contraception and underscores the multimerization of IgG as a promising strategy to enhance antibody effector functions based on immune exclusion.

Engineering monoclonal antibody-based contraception and multipurpose prevention technologies†.

Sexually transmitted infections are highly prevalent, and over 40% of pregnancies are unplanned. We are producing new antibody-based multipurpose prevention technology products to address these problems and fill an unmet need in female reproductive health. We used a Nicotiana platform to manufacture monoclonal antibodies against two prevalent sexually transmitted pathogens, HIV-1 and HSV-2, and incorporated them into a vaginal film (MB66) for preclinical and Phase 1 clinical testing. These tests are now complete and indicate that MB66 is effective and safe in women. We are now developing an antisperm monoclonal antibody to add contraceptive efficacy to this product. The antisperm antibody, H6-3C4, originally isolated by Shinzo Isojima from the blood of an infertile woman, recognizes a carbohydrate epitope on CD52g, a glycosylphosphatidylinositol-anchored glycoprotein found in abundance on the surface of human sperm. We engineered the antibody for production in Nicotiana; the new antibody which we call "human contraception antibody," effectively agglutinates sperm at concentrations >10 µg/ml and maintains activity under a variety of physiological conditions. We are currently seeking regulatory approval for a Phase 1 clinical trial, which will include safety and "proof of principle" efficacy endpoints. Concurrently, we are working with new antibody production platforms to bring the costs down, innovative antibody designs that may produce more effective second-generation antibodies, and delivery systems to provide extended protection.

The cervicovaginal mucus barrier to HIV-1 is diminished in bacterial vaginosis.

Bacterial vaginosis (BV), a condition in which the vaginal microbiota consists of community of obligate and facultative anaerobes rather than dominated by a single species of Lactobacillus, affects ~30% of women in the US. Women with BV are at 60% increased risk for HIV acquisition and are 3-times more likely to transmit HIV to an uninfected partner. As cervicovaginal mucus (CVM) is the first line of defense against mucosal pathogens and the home of the resident vaginal microbiota, we hypothesized the barrier function of CVM to HIV may be diminished in BV. Here, we characterized CVM properties including pH, lactic acid content, and Nugent score to correlate with the microbiota community composition, which was confirmed by 16S rDNA sequencing on a subset of samples. We then quantified the mobility of fluorescently-labeled HIV virions and nanoparticles to characterize the structural and adhesive barrier properties of CVM. Our analyses included women with Nugent scores categorized as intermediate (4-6) and BV (7-10), women that were either symptomatic or asymptomatic, and a small group of women before and after antibiotic treatment for symptomatic BV. Overall, we found that HIV virions had significantly increased mobility in CVM from women with BV compared to CVM from women with Lactobacillus crispatus-dominant microbiota, regardless of whether symptoms were present. We confirmed using nanoparticles and scanning electron microscopy that the impaired barrier function was due to reduced adhesive barrier properties without an obvious degradation of the physical CVM pore structure. We further confirmed a similar increase in HIV mobility in CVM from women with Lactobacillus iners-dominant microbiota, the species most associated with transitions to BV and that persists after antibiotic treatment for BV. Our findings advance the understanding of the protective role of mucus and highlight the interplay between vaginal microbiota and the innate barrier function mucus.

A Randomized, Controlled Trial of Ebola Virus Disease Therapeutics.

BACKGROUND: Although several experimental therapeutics for Ebola virus disease (EVD) have been developed, the safety and efficacy of the most promising therapies need to be assessed in the context of a randomized, controlled trial. METHODS: We conducted a trial of four investigational therapies for EVD in the Democratic Republic of Congo, where an outbreak began in August 2018. Patients of any age who had a positive result for Ebola virus RNA on reverse-transcriptase-polymerase-chain-reaction assay were enrolled. All patients received standard care and were randomly assigned in a 1:1:1:1 ratio to intravenous administration of the triple monoclonal antibody ZMapp (the control group), the antiviral agent remdesivir, the single monoclonal antibody MAb114, or the triple monoclonal antibody REGN-EB3. The REGN-EB3 group was added in a later version of the protocol, so data from these patients were compared with those of patients in the ZMapp group who were enrolled at or after the time the REGN-EB3 group was added (the ZMapp subgroup). The primary end point was death at 28 days. RESULTS: A total of 681 patients were enrolled from November 20, 2018, to August 9, 2019, at which time the data and safety monitoring board recommended that patients be assigned only to the MAb114 and REGN-EB3 groups for the remainder of the trial; the recommendation was based on the results of an interim analysis that showed superiority of these groups to ZMapp and remdesivir with respect to mortality. At 28 days, death had occurred in 61 of 174 patients (35.1%) in the MAb114 group, as compared with 84 of 169 (49.7%) in the ZMapp group (P = 0.007), and in 52 of 155 (33.5%) in the REGN-EB3 group, as compared with 79 of 154 (51.3%) in the ZMapp subgroup (P = 0.002). A shorter duration of symptoms before admission and lower baseline values for viral load and for serum creatinine and aminotransferase levels each correlated with improved survival. Four serious adverse events were judged to be potentially related to the trial drugs. CONCLUSIONS: Both MAb114 and REGN-EB3 were superior to ZMapp in reducing mortality from EVD. Scientifically and ethically sound clinical research can be conducted during disease outbreaks and can help inform the outbreak response. (Funded by the National Institute of Allergy and Infectious Diseases and others; PALM ClinicalTrials.gov number, NCT03719586.).

Vaginal pH measured in vivo: lactobacilli determine pH and lactic acid concentration.

BACKGROUND: Lactic acid (protonated lactate) has broad antimicrobial activity. Vaginal lactobacilli produce lactic acid, and are known to confer protection against reproductive tract infections when they are predominant in the vaginal microbiota. Using novel ex vivo methods, we showed that cervicovaginal fluid (CVF) from women with a predominantly lactobacilli-morphotype microbiota contains significantly more lactic acid than previously thought, sufficient to inactivate reproductive tract pathogens. Here, we measured vaginal pH in vivo in 20 women with a predominantly lactobacilli-morphotype (low Nugent score) microbiota. We also investigated the in vitro production of protons (as hydrogen ions) and lactate by vaginal lactobacilli. RESULTS: The average vaginal pH in these women was 3.80 ± 0.20, and the average lactate concentration was 0.79% ± 0.22% w/v, with pH and lactate concentration tightly correlated for each sample. In vitro, lactobacilli cultured from these CVF samples reached an average pH of 3.92 ± 0.22, but the average lactate concentration was only 0.14% ± 0.06% w/v, approximately five-fold less than in the corresponding CVF samples. When the pH of the cultures was raised, lactate and hydrogen ion production resumed, indicating that production of lactate and hydrogen ions by vaginal lactobacilli is limited primarily by their sensitivity to hydrogen ion concentration (low pH) not lactate concentration. CONCLUSIONS: Some vaginal lactobacilli cultures have a lower limiting pH than others, and limiting pHs in vitro showed good correlation with pHs measured in vivo. The limiting pH of the lactobacilli predominant in a woman's vaginal microbiota seems critical in determining the concentration of antimicrobial lactic acid protecting her.

Anti-HIV-1 Activity of Lactic Acid in Human Cervicovaginal Fluid.

Women of reproductive age with a Lactobacillus-dominated vaginal microbiota have a reduced risk of acquiring and transmitting HIV and a vaginal pH of ~4 due to the presence of ~1% (wt/vol) lactic acid. While lactic acid has potent HIV virucidal activity in vitro, whether lactic acid present in the vaginal lumen inactivates HIV has not been investigated. Here we evaluated the anti-HIV-1 activity of native, minimally diluted cervicovaginal fluid obtained from women of reproductive age (n = 20) with vaginal microbiota dominated by Lactobacillus spp. Inhibition of HIVBa-L was significantly associated with the protonated form of lactic acid in cervicovaginal fluid. The HIVBa-L inhibitory activity observed in the <3-kDa acidic filtrate was similar to that of the corresponding untreated native cervicovaginal fluid as well as that of clarified neat cervicovaginal fluid subjected to protease digestion. These ex vivo studies indicate that protonated lactic acid is a major anti-HIV-1 metabolite present in acidic cervicovaginal fluid, suggesting a potential role in reducing HIV transmission by inactivating virus introduced or shed into the cervicovaginal lumen.IMPORTANCE The Lactobacillus-dominated vaginal microbiota is associated with a reduced risk of acquiring and transmitting HIV and other sexually transmitted infections (STIs). Lactic acid is a major organic acid metabolite produced by lactobacilli that acidifies the vagina and has been reported to have inhibitory activity in vitro against bacterial, protozoan, and viral STIs, including HIV infections. However, the anti-HIV properties of lactic acid in native vaginal lumen fluids of women colonized with Lactobacillus spp. have not yet been established. Our study, using native cervicovaginal fluid from women, found that potent and irreversible anti-HIV-1 activity is significantly associated with the concentration of the protonated (acidic, uncharged) form of lactic acid. This work advances our understanding of the mechanisms by which vaginal microbiota modulate HIV susceptibility and could lead to novel strategies to prevent women from acquiring HIV or transmitting the virus during vaginal intercourse and vaginal birth.

Attitudes and perceptions towards novel objective measures of ARV-based vaginal ring use: Results from a global stakeholder survey.

Results of recent microbicide and pre-exposure prophylaxis clinical trials have shown adherence to be a significant challenge with new HIV prevention technologies. As the vaginal ring containing dapivirine moves into two open label follow-on studies (HOPE/MTN-025 and DREAM) and other antiretroviral-based and multi-purpose prevention technology ring products advance through the development pipeline, there is a need for more accurate and reliable measures of adherence to microbicide ring products. We previously conducted a comprehensive landscape analysis to identify new technologies that could be applied to adherence measurement of vaginal rings containing antiretrovirals. To explore attitudes and perceptions towards the approaches that we identified, we conducted a survey of stakeholders with experience and expertise in microbicide and HIV prevention clinical trials. From May to July 2015 an electronic survey was distributed via email to 894 stakeholders; a total of 206 eligible individuals responded to at least one question and were included in the data analysis. Survey respondents were presented with various objective measures and asked about their perceived acceptability to trial participants, feasibility of implementation by study staff, usefulness for measuring adherence and ethical concerns. Methods that require no additional input from the participant and require no modifications to the existing ring product (i.e., measurement of residual drug or excipient, or a vaginal analyte that enters the ring) were viewed as being more acceptable to trial participants and more feasible to implement in the field. Respondents saw value in using objective measures to provide real-time feedback on adherence. However, approaches that involve unannounced home visits for sample collection or spot checks of ring use, which could provide significant value to adherence feedback efforts, were met with skepticism. Additional research on the acceptability of these methods to potential trial participants and trial staff is recommended.

Comparison of the vaginal environment in rhesus and cynomolgus macaques pre- and post-lactobacillus colonization.

BACKGROUND: Rhesus and cynomologus macaques are valuable animal models for the study of human immunodeficiency virus (HIV) prevention strategies. However, for such studies focused on the vaginal route of infection, differences in vaginal environment may have deterministic impact on the outcome of such prevention, providing the rationale for this study. METHODS: We tested the vaginal environment of rhesus and cynomolgus macaques longitudinally to characterize the normal microflora based on Nugent scores and pH. This evaluation was extended after colonization of the vaginal space with Lactobacilli in an effort to recreate NHP models representing the healthy human vaginal environment. RESULTS AND CONCLUSION: Nugent scores and pH differed significantly between species, although data from both species were suggestive of stable bacterial vaginosis. Colonization with Lactobacilli was successful in both species leading to lower Nugent score and pH, although rhesus macaques appeared better able to sustain Lactobacillus spp over time.

Pharmacokinetics and Preliminary Safety of Pod-Intravaginal Rings Delivering the Monoclonal Antibody VRC01-N for HIV Prophylaxis in a Macaque Model.

The broadly neutralizing antibody (bNAb) VRC01, capable of neutralizing 91% of known human immunodeficiency virus type 1 (HIV-1) isolates in vitro, is a promising candidate microbicide for preventing sexual HIV infection when administered topically to the vagina; however, accessibility to antibody-based prophylactic treatment by target populations in sub-Saharan Africa and other underdeveloped regions may be limited by the high cost of conventionally produced antibodies and the limited capacity to manufacture such antibodies. Intravaginal rings of the pod design (pod-IVRs) delivering Nicotiana-manufactured VRC01 (VRC01-N) over a range of release rates have been developed. The pharmacokinetics and preliminary safety of VRC01-N pod-IVRs were evaluated in a rhesus macaque model. The devices sustained VRC01-N release for up to 21 days at controlled rates, with mean steady-state VRC01-N levels in vaginal fluids in the range of 102 to 103 µg g-1 being correlated with in vitro release rates. No adverse safety indications were observed. These findings indicate that pod-IVRs are promising devices for the delivery of the candidate topical microbicide VRC01-N against HIV-1 infection and merit further preclinical evaluation.

Anti-PEG antibodies alter the mobility and biodistribution of densely PEGylated nanoparticles in mucus.

UNLABELLED: Antibodies that specifically bind polyethylene glycol (PEG) can lead to rapid elimination of PEGylated therapeutics from the systemic circulation. We have recently shown that virus-binding IgG can immobilize viruses in mucus via multiple low-affinity crosslinks between IgG and mucins. However, it remains unclear whether anti-PEG antibodies in mucus may also alter the penetration and consequently biodistribution of PEGylated nanoparticles delivered to mucosal surfaces. We found that both anti-PEG IgG and IgM can readily bind nanoparticles that were densely coated with PEG polymer to minimize adhesive interactions with mucus constituents. Addition of anti-PEG IgG and IgM into mouse cervicovaginal mucus resulted in extensive trapping of mucus-penetrating PEGylated nanoparticles, with the fraction of mobile particles reduced from over 95% to only 34% and 7% with anti-PEG IgG and IgM, respectively. Surprisingly, we did not observe significant agglutination induced by either antibody, suggesting that particle immobilization is caused by adhesive crosslinks between mucin fibers and IgG or IgM bound to individual nanoparticles. Importantly, addition of corresponding control antibodies did not slow the PEGylated nanoparticles, confirming anti-PEG antibodies specifically bound to and trapped the PEGylated nanoparticles. Finally, we showed that trapped PEGylated nanoparticles remained largely in the luminal mucus layer of the mouse vagina even when delivered in hypotonic formulations that caused untrapped particles to be drawn by the flow of water (advection) through mucus all the way to the epithelial surface. These results underscore the potential importance of elucidating mucosal anti-PEG immune responses for PEGylated therapeutics and biomaterials applied to mucosal surfaces. STATEMENT OF SIGNIFICANCE: PEG, generally considered a 'stealth' polymer, is broadly used to improve the circulation times and therapeutic efficacy of nanomedicines. Nevertheless, there is increasing scientific evidence that demonstrates both animals and humans can generate PEG-specific antibodies. Here, we show that anti-PEG IgG and IgM can specifically immobilize otherwise freely diffusing PEG-coated nanoparticles in fresh vaginal mucus gel ex vivo by crosslinking nanoparticles to the mucin mesh, and consequently prevent PEG-coated nanoparticles from accessing the vaginal epithelium in vivo. Given the increasing use of PEG coatings to enhance nanoparticle penetration of mucosal barriers, our findings demonstrate that anti-PEG immunity may be a potential concern not only for systemic drug delivery but also for mucosal drug delivery.

Biomarkers and biometric measures of adherence to use of ARV-based vaginal rings.

INTRODUCTION: Poor adherence to product use has been observed in recent trials of antiretroviral (ARV)-based oral and vaginal gel HIV prevention products, resulting in an inability to determine product efficacy. The delivery of microbicides through vaginal rings is widely perceived as a way to achieve better adherence but vaginal rings do not eliminate the adherence challenges exhibited in clinical trials. Improved objective measures of adherence are needed as new ARV-based vaginal ring products enter the clinical trial stage. METHODS: To identify technologies that have potential future application for vaginal ring adherence measurement, a comprehensive literature search was conducted that covered a number of biomedical and public health databases, including PubMed, Embase, POPLINE and the Web of Science. Published patents and patent applications were also searched. Technical experts were also consulted to gather more information and help evaluate identified technologies. Approaches were evaluated as to feasibility of development and clinical trial implementation, cost and technical strength. RESULTS: Numerous approaches were identified through our landscape analysis and classified as either point measures or cumulative measures of vaginal ring adherence. Point measurements are those that give a measure of adherence at a particular point in time. Cumulative measures attempt to measure ring adherence over a period of time. DISCUSSION: Approaches that require modifications to an existing ring product are at a significant disadvantage, as this will likely introduce additional regulatory barriers to the development process and increase manufacturing costs. From the point of view of clinical trial implementation, desirable attributes would be high acceptance by trial participants, and little or no additional time or training requirements on the part of participants or clinic staff. We have identified four promising approaches as being high priority for further development based on the following measurements: intracellular drug levels, drug levels in hair, the accumulation of a vaginal analyte that diffuses into the ring, and the depletion of an intrinsic ring constituent. CONCLUSIONS: While some approaches show significant promise over others, it is recommended that a strategy of using complementary biometric and behavioural approaches be adopted to best understand participants' adherence to ARV-based ring products in clinical trials.

Enema ion compositions for enhancing colorectal drug delivery.

Delivering drugs to the colorectum by enema has advantages for treating or preventing both local and systemic diseases. However, the properties of the enema itself are not typically exploited for improving drug delivery. Sodium ions are actively pumped out of the lumen of the colon, which is followed by osmotically-driven water absorption, so we hypothesized that this natural mechanism could be exploited to drive nanoparticles and drugs to the colorectal tissue surface. Here, we report that sodium-based, absorption-inducing (hypotonic) enemas rapidly transport hydrophilic drugs and non-mucoadhesive, mucus penetrating nanoparticles (MPP), deep into the colorectal folds to reach virtually the entire colorectal epithelial surface. In contrast, isotonic and secretion-inducing (hypertonic) vehicles led to non-uniform, poor surface coverage. Sodium-based enemas induced rapid fluid absorption even when moderately hyper-osmolal (~350 mOsm) compared to blood (~300 mOsm), which suggests that active sodium absorption plays a key role in osmosis-driven fluid uptake. We then used tenofovir, an antiretroviral drug in clinical trials for preventing HIV, to test the effects of enema composition on local and systemic drug delivery. We found that strongly hypotonic and hypertonic enemas caused rapid systemic drug uptake, whereas moderately hypotonic enemas with ion compositions similar to feces resulted in high local tissue levels with minimal systemic drug exposure. Similarly, moderately hypotonic enemas provided improved local drug retention in colorectal tissue, whereas hypertonic and isotonic enemas provided markedly reduced drug retention in colorectal tissue. Lastly, we found that moderately hypotonic enema formulations caused little to no detectable epithelial damage, while hypertonic solutions caused significant damage, including epithelial sloughing; the epithelial damage caused increased systemic drug absorption and penetration of MPP into colorectal tissue, a potential advantage in certain drug delivery applications. In summary, we illustrate that enema composition can be adjusted to maximize local versus systemic drug delivery, and that mildly hypotonic, sodium-based vehicles can provide uniform drug and MPP delivery in the colon that maximizes local drug concentrations.

Cell-associated transmission of HIV type 1 and other lentiviruses in small-animal models.

Small-animal models of lentivirus transmission have repeatedly demonstrated transmission by cell-associated virus via vaginal, rectal, and oral routes. The earliest experiments were in the cat/feline immunodeficiency virus model, followed a decade later by successful vaginal transmission of cell-associated human immunodeficiency virus (HIV) in mice bearing transplanted human immune cells. After early unsuccessful attempts at cell-associated transmission in nonhuman primates, renewed investigation in diverse primate models has now confirmed the findings from the cat and humanized mouse models. Improvements in humanized mouse models have made them the preferred small-animal models to study HIV mucosal transmission. They provide complementary systems to nonhuman primate models to aid in the elucidation of the many remaining questions on the mechanism of and means to prevent both cell-associated and cell-free HIV transmission across mucosal barriers.