A Label-free Optical Biosensor-Based Point-of-Care Test for the Rapid Detection of Monkeypox Virus.

Diagnostic approaches that combine the high sensitivity and specificity of laboratory-based digital detection with the ease of use and affordability of point-of-care (POC) technologies could revolutionize disease diagnostics. This is especially true in infectious disease diagnostics, where rapid and accurate pathogen detection is critical to curbing the spread of disease. We have pioneered an innovative label-free digital detection platform that utilizes Interferometric Reflectance Imaging Sensor (IRIS) technology. IRIS leverages light interference from an optically transparent thin film, eliminating the need for complex optical resonances to enhance the signal by harnessing light interference and the power of signal averaging in shot-noise-limited operation to achieve virtually unlimited sensitivity. In our latest work, we have further improved our previous 'Single-Particle' IRIS (SP-IRIS) technology by allowing the construction of the optical signature of target nanoparticles (whole virus) from a single image. This new platform, 'Pixel-Diversity' IRIS (PD-IRIS), eliminated the need for z-scan acquisition, required in SP-IRIS, a time-consuming and expensive process, and made our technology more applicable to POC settings. Using PD-IRIS, we quantitatively detected the Monkeypox virus (MPXV), the etiological agent for Monkeypox (Mpox) infection. MPXV was captured by anti-A29 monoclonal antibody (mAb 69-126-3) on Protein G spots on the sensor chips and were detected at a limit-of-detection (LOD) - of 200 PFU/ml (∼3.3 attomolar). PD-IRIS was superior to the laboratory-based ELISA (LOD - 1800 PFU/mL) used as a comparator. The specificity of PD-IRIS in MPXV detection was demonstrated using Herpes simplex virus, type 1 (HSV-1), and Cowpox virus (CPXV). This work establishes the effectiveness of PD-IRIS and opens possibilities for its advancement in clinical diagnostics of Mpox at POC. Moreover, PD-IRIS is a modular technology that can be adapted for the multiplex detection of pathogens for which high-affinity ligands are available that can bind their surface antigens to capture them on the sensor surface.

Ocular Mpox in a Breastfeeding Healthcare Provider.

A healthcare provider unknowingly treated a patient with mpox and subsequently developed ocular mpox without rash. She breastfed during illness; her infant was not infected. This report addresses 3 challenges in mpox management and control: diagnosis in the absence of rash, exposures in healthcare settings, and management of lactating patients.

Minimally Invasive Blood Collection for an Mpox Serosurvey among People Experiencing Homelessness.

BACKGROUND: People experiencing homelessness (PEH) are underrepresented in public health and clinical research. Study methods that can improve participation by this group are needed. METHODS: In late 2022, the Centers for Disease Control and Prevention conducted an mpox serological survey using venipuncture among PEH in San Francisco, California. Blood collection by a minimally invasive device was offered if venipuncture was not possible or preferred. Participants who had a successful blood draw using the device were asked about device acceptability. RESULTS: Of the 209 successful blood collections, 137 (66%) were among participants who underwent venipuncture and 72 (34%) were among participants who used the device. Use of the device increased overall blood collection participation by 53%. Participants reported high acceptability and preference for the device over venipuncture. CONCLUSIONS: Minimally invasive blood collection devices may increase participation and representation of PEH in serosurveys.

Serologic responses to the MVA-based JYNNEOS mpox vaccine in a cohort of participants from the District of Columbia (D.C.).

We assessed early antibody responses after two doses of JYNNEOS (IMVANEX) mpox vaccine in the District of Columbia (D.C.) in persons at high risk for mpox without characteristic lesions or rash. Participants with PCR mpox negative specimens (oral swab, blood, and/or rectal swab) on the day of receipt of the first vaccine dose and who provided a baseline (day 0) serum sample and at least one serum sample at âˆ¼28, ∼42-56 days, or 180 days post vaccination were included in this analysis. Orthopoxvirus (OPXV)-specific IgG and IgM ELISAs and neutralizing antibody titers were performed, and longitudinal serologic responses were examined. Based on participants' IgG and IgM antibody levels at baseline, they were categorized as naïve or non-naïve. Linear mixed effects regression models were conducted to determine if IgG antibody response over time varied by age, sex, HIV status, and route of administration for both naïve and non-naïve participants. Among both naïve and non-naïve participants IgG seropositivity rates increased until day 42-56, with 89.4 % of naïve and 92.1 % of non-naïve participants having detectable IgG antibodies. The proportion of naive participants with detectable IgG antibodies declined by day 180 (67.7 %) but remained high among non-naïve participants (94.4 %). Neutralizing antibody titers displayed a similar pattern, increasing initially post vaccination but declining by day 180 among naïve participants. There were no significant serologic response differences by age, sex, or HIV status. Serologic response did vary by route of vaccine administration, with those receiving a combination of intradermal and subcutaneous doses displaying significantly higher IgG values than those receiving both doses intradermally. These analyses provide initial insights into the immunogenicity of a two-dose JYNNEOS PEP regimen in individuals at high risk of mpox exposure in the United States.

Serological Evidence of Mpox Virus Infection During Peak Mpox Transmission in New York City, July to August 2022.

BACKGROUND: The extent to which infections may have been undetected in an epicenter of the 2022 mpox outbreak is unknown. METHODS: A serosurvey (July and August 2022) assessed the seroprevalence and correlates of mpox infection among a diverse sample of asymptomatic patients with no prior mpox diagnoses and no known histories of smallpox or mpox vaccination. We present seropositivity stratified by participant characteristics collected via survey. RESULTS: Two-thirds of 419 participants were cismen (281 of 419), of whom 59.1% (166 of 281) reported sex with men (MSM). The sample also included 109 ciswomen and 28 transgender/gender nonconforming/nonbinary individuals. Overall seroprevalence was 6.4% (95% confidence interval [CI], 4.1%-8.8%); 3.7% among ciswomen (95% CI, 1.0%-9.1%), 7.0% among cismen with only ciswomen partners (95% CI, 2.0%-11.9%), and 7.8% among MSM (95% CI, 3.7%-11.9%). There was little variation in seroprevalence by race/ethnicity, age group, HIV status, or number of recent sex partners. No participants who reported close contact with mpox cases were seropositive. Among participants without recent mpox-like symptoms, 6.3% were seropositive (95% CI, 3.6%-9.0%). CONCLUSIONS: Approximately 1 in 15 vaccine-naive people in our study had antibodies to mpox during the height of the NYC outbreak, indicating the presence of asymptomatic infections that could contribute to ongoing transmission.

Investigation of an mpox outbreak affecting many vaccinated persons in Chicago, IL-March 2023-June 2023.

BACKGROUND: After months of few mpox cases, an increased number of cases were reported in Chicago during May 2023; predominantly among fully vaccinated patients. We investigated the outbreak scope, differences between vaccinated and unvaccinated patients, and hypotheses for monkeypox virus (MPXV) infection after vaccination. METHODS: We interviewed patients and reviewed medical records to assess demographic, behavioral, and clinical characteristics, mpox vaccine status, and vaccine administration routes. We evaluated serum antibody levels after infection and compared patient viral genomes with MPXV sequences in available databases. We discussed potential vaccine compromise with partners who manufactured, handled, and administered vaccine associated with breakthrough infections. RESULTS: During March 18-June 27, 2023, we identified 49 mpox cases; 57% of these mpox patients were fully vaccinated (FV). FV patients received both JYNNEOS doses subcutaneously (57%), intradermally (7%), or via heterologous administration (36%). FV patients had more median sex partners (3, IQR=1-4) versus not fully vaccinated (NFV) patients (1, IQR=1-2). Thirty-six of 37 sequenced specimens belonged to lineage B.1.20 of clade IIb MPXV, which did not demonstrate any amino acid changes relative to B.1, the predominant lineage from May 2022. Vaccinated patients demonstrated expected humoral antibody responses; none were hospitalized. No vaccine storage excursions were identified. Approximately 63% of people at risk for mpox in Chicago were FV during this period. CONCLUSIONS: Our investigation indicated cases were likely due to frequent behaviors associated with mpox transmission, even with relatively high vaccine effectiveness and vaccine coverage. Cases after vaccination might occur in similar populations.

How the Orthodox Features of Orthopoxviruses Led to an Unorthodox Mpox Outbreak: What We've Learned, and What We Still Need to Understand.

Orthopoxviruses have repeatedly confounded expectations in terms of the clinical illness they cause and their patterns of spread. Monkeypox virus (MPXV), originally characterized in the late 1950s during outbreaks among captive primates, has been recognized since the 1970s to cause human disease (mpox) in West and Central Africa, where interhuman transmission has largely been associated with nonsexual, close physical contact. In May 2022, a focus of MPXV transmission was detected, spreading among international networks of gay, bisexual, and other men who have sex with men. The outbreak grew in both size and geographic scope, testing the strength of preparedness tools and public health science alike. In this article we consider what was known about mpox before the 2022 outbreak, what we learned about mpox during the outbreak, and what continued research is needed to ensure that the global public health community can detect, and halt further spread of this disease threat.

Prevalence of Undiagnosed Monkeypox Virus Infections during Global Mpox Outbreak, United States, June-September 2022.

Since May 2022, mpox has been identified in 108 countries without endemic disease; most cases have been in gay, bisexual, or other men who have sex with men. To determine number of missed cases, we conducted 2 studies during June-September 2022: a prospective serologic survey detecting orthopoxvirus antibodies among men who have sex with men in San Francisco, California, and a retrospective monkeypox virus PCR testing of swab specimens submitted for other infectious disease testing among all patients across the United States. The serosurvey of 225 participants (median age 34 years) detected 18 (8.0%) who were orthopoxvirus IgG positive and 3 (1.3%) who were also orthopoxvirus IgM positive. The retrospective PCR study of 1,196 patients (median age 30 years; 54.8% male) detected 67 (5.6%) specimens positive for monkeypox virus. There are likely few undiagnosed cases of mpox in regions where sexual healthcare is accessible and patient and clinician awareness about mpox is increased.

Monkeypox virus-infected individuals mount comparable humoral immune responses as Smallpox-vaccinated individuals.

In early 2022, a cluster of monkeypox virus (MPXV) infection (mpox) cases were identified within the UK with no prior travel history to MPXV-endemic regions. Subsequently, case numbers exceeding 80,000 were reported worldwide, primarily affecting gay, bisexual, and other men who have sex with men (GBMSM). Public health agencies worldwide have offered the IMVANEX Smallpox vaccination to these individuals at high-risk to provide protection and limit the spread of MPXV. We have developed a comprehensive array of ELISAs to study poxvirus-induced antibodies, utilising 24 MPXV and 3 Vaccinia virus (VACV) recombinant antigens. Panels of serum samples from individuals with differing Smallpox-vaccine doses and those with prior MPXV infection were tested on these assays, where we observed that one dose of Smallpox vaccination induces a low number of antibodies to a limited number of MPXV antigens but increasing with further vaccination doses. MPXV infection induced similar antibody responses to diverse poxvirus antigens observed in Smallpox-vaccinated individuals. We identify MPXV A27 as a serological marker of MPXV-infection, whilst MPXV M1 (VACV L1) is likely IMVANEX-specific. Here, we demonstrate analogous humoral antigen recognition between both MPXV-infected or Smallpox-vaccinated individuals, with binding to diverse yet core set of poxvirus antigens, providing opportunities for future vaccine (e.g., mRNA) and therapeutic (e.g., mAbs) design.

Mosquito Repellent Efficacy of Australian Blue Cypress Callitris intratropica Essential Oil and a Topical Formulation under Laboratory and Field Conditions.

Mosquito repellents are important for personal protection against nuisance and potentially infectious mosquito bites. Repellent activity of Australian blue cypress essential oil (EO) and a commercially formulated skin lotion containing blue cypress EO (topical formulation) were compared with 20% DEET (N, N-diethyl-3 toluamide) against mosquitoes under laboratory and field conditions in North Queensland, Australia. On a volunteer's forearm, 1 mL of candidate material was applied to approximately 600 cm2 of exposed skin. When blue cypress EO was applied at various concentrations (0.5%-10.5%), it did not fully prevent mosquito landing or biting. However, a dose-dependent increase, approaching 80% protection, was observed at high EO concentrations. On the basis of these results, three concentrations (5%, 10%, and 20%) of blue cypress EO were selected for complete protection time (CPT) experiments. Topical formulation (undiluted) was also included in CPT experiments. Although some protection was afforded, mosquito landing/probing were still recorded immediately after application for both blue cypress EO and its topical formulation. Specifically, protection declined for blue cypress EO from 80% to 70% (laboratory) and from 93% to 50% (field) within 1 hour. For topical formulation, protection declined from 85% to 75% in the laboratory and from 63% to 50% in the field. In comparison, DEET maintained a 100% protection throughout the testing period of up to 1 h, and there was no landing/probing observed in volunteers who had applied DEET. To conclude, both blue cypress products provided some protection against mosquito bites, which decreased soon after application.

An agonistic anti-signal regulatory protein α antibody for chronic inflammatory diseases.

Signal regulatory protein (SIRPα) is an immune inhibitory receptor expressed by myeloid cells to inhibit immune cell phagocytosis, migration, and activation. Despite the progress of SIRPα and CD47 antagonist antibodies to promote anti-cancer immunity, it is not yet known whether SIRPα receptor agonism could restrain excessive autoimmune tissue inflammation. Here, we report that neutrophil- and monocyte-associated genes including SIRPA are increased in inflamed tissue biopsies from patients with rheumatoid arthritis and inflammatory bowel diseases, and elevated SIRPA is associated with treatment-refractory ulcerative colitis. We next identify an agonistic anti-SIRPα antibody that exhibits potent anti-inflammatory effects in reducing neutrophil and monocyte chemotaxis and tissue infiltration. In preclinical models of arthritis and colitis, anti-SIRPα agonistic antibody ameliorates autoimmune joint inflammation and inflammatory colitis by reducing neutrophils and monocytes in tissues. Our work provides a proof of concept for SIRPα receptor agonism for suppressing excessive innate immune activation and chronic inflammatory disease treatment.

A systems biology approach uncovers novel disease mechanisms in age-related macular degeneration.

Age-related macular degeneration (AMD) is a leading cause of blindness, affecting 200 million people worldwide. To identify genes that could be targeted for treatment, we created a molecular atlas at different stages of AMD. Our resource is comprised of RNA sequencing (RNA-seq) and DNA methylation microarrays from bulk macular retinal pigment epithelium (RPE)/choroid of clinically phenotyped normal and AMD donor eyes (n = 85), single-nucleus RNA-seq (164,399 cells), and single-nucleus assay for transposase-accessible chromatin (ATAC)-seq (125,822 cells) from the retina, RPE, and choroid of 6 AMD and 7 control donors. We identified 23 genome-wide significant loci differentially methylated in AMD, over 1,000 differentially expressed genes across different disease stages, and an AMD Müller state distinct from normal or gliosis. Chromatin accessibility peaks in genome-wide association study (GWAS) loci revealed putative causal genes for AMD, including HTRA1 and C6orf223. Our systems biology approach uncovered molecular mechanisms underlying AMD, including regulators of WNT signaling, FRZB and TLE2, as mechanistic players in disease.

Clinical characterization and placental pathology of mpox infection in hospitalized patients in the Democratic Republic of the Congo.

We describe the results of a prospective observational study of the clinical natural history of human monkeypox (mpox) virus (MPXV) infections at the remote L'Hopital General de Reference de Kole (Kole hospital), the rainforest of the Congo River basin of the Democratic Republic of the Congo (DRC) from March 2007 until August 2011. The research was conducted jointly by the Institute National de Recherche Biomedical (INRB) and the US Army Medical Research Institute of Infectious Diseases (USAMRIID). The Kole hospital was one of the two previous WHO Mpox study sites (1981-1986). The hospital is staffed by a Spanish Order of Catholic Nuns from La Congregation Des Soeurs Missionnaires Du Christ Jesus including two Spanish physicians, who were members of the Order as well, were part of the WHO study on human mpox. Of 244 patients admitted with a clinical diagnosis of MPXV infection, 216 were positive in both the Pan-Orthopox and MPXV specific PCR. The cardinal observations of these 216 patients are summarized in this report. There were three deaths (3/216) among these hospitalized patients; fetal death occurred in 3 of 4 patients who were pregnant at admission, with the placenta of one fetus demonstrating prominent MPXV infection of the chorionic villi. The most common complaints were rash (96.8%), malaise (85.2%), sore throat (78.2%), and lymphadenopathy/adenopathy (57.4%). The most common physical exam findings were mpox rash (99.5%) and lymphadenopathy (98.6%). The single patient without the classic mpox rash had been previously vaccinated against smallpox. Age group of less than 5 years had the highest lesion count. Primary household cases tended to have higher lesion counts than secondary or later same household cases. Of the 216 patients, 200 were tested for IgM & IgG antibodies (Abs) to Orthopoxviruses. All 200 patients had anti-orthopoxvirus IgG Abs; whereas 189/200 were positive for IgM. Patients with hypoalbuminemia had a high risk of severe disease. Patients with fatal disease had higher maximum geometric mean values than survivors for the following variables, respectively: viral DNA in blood (DNAemia); maximum lesion count; day of admission mean AST and ALT.

Possible Undetected Mpox Infection Among Persons Accessing Homeless Services and Staying in Encampments - San Francisco, California, October-November 2022.

Monkeypox (mpox) is a disease caused by an Orthopoxvirus. The 2022 multinational outbreak, which began in May 2022, has spread primarily by close skin-to-skin contact, including through sexual contact. Persons experiencing homelessness have been disproportionately affected by severe mpox (1). However, mpox prevalence and transmission pathways among persons experiencing homelessness are not known, and persons experiencing homelessness have not been specifically recommended to receive mpox vaccine during the 2022 outbreak (2,3). During October 25-November 3, 2022, a CDC field team conducted an orthopoxvirus seroprevalence survey among persons accessing homeless services or staying in encampments, shelters, or permanent supportive housing in San Francisco, California that had noted at least one case of mpox or served populations at risk. During field team visits to 16 unique sites, 209 participants completed a 15-minute survey and provided a blood specimen. Among 80 participants aged <50 years who did not report smallpox or mpox vaccination or previous mpox infection, two (2.5%) had detectable antiorthopoxvirus immunoglobulin (Ig) G antibody. Among 73 participants who did not report mpox vaccination or previous mpox infection and who were tested for IgM, one (1.4%) had detectable antiorthopoxvirus IgM. Together, these results suggest that three possible undetected mpox infections occurred among a sample of persons experiencing homelessness, highlighting the need to ensure that community outreach and prevention interventions, such as vaccination, are accessible to this population.

Design and Optimization of a Monkeypox virus Specific Serological Assay.

Monkeypox virus (MPXV), a member of the Orthopoxvirus (OPXV) genus, is a zoonotic virus, endemic to central and western Africa that can cause smallpox-like symptoms in humans with fatal outcomes in up to 15% of patients. The incidence of MPXV infections in the Democratic Republic of the Congo, where the majority of cases have occurred historically, has been estimated to have increased as much as 20-fold since the end of smallpox vaccination in 1980. Considering the risk global travel carries for future disease outbreaks, accurate epidemiological surveillance of MPXV is warranted as demonstrated by the recent Mpox outbreak, where the majority of cases were occurring in non-endemic areas. Serological differentiation between childhood vaccination and recent infection with MPXV or other OPXVs is difficult due to the high level of conservation within OPXV proteins. Here, a peptide-based serological assay was developed to specifically detect exposure to MPXV. A comparative analysis of immunogenic proteins across human OPXVs identified a large subset of proteins that could potentially be specifically recognized in response to a MPXV infection. Peptides were chosen based upon MPXV sequence specificity and predicted immunogenicity. Peptides individually and combined were screened in an ELISA against serum from well-characterized Mpox outbreaks, vaccinee sera, and smallpox sera collected prior to eradication. One peptide combination was successful with ~86% sensitivity and ~90% specificity. The performance of the assay was assessed against the OPXV IgG ELISA in the context of a serosurvey by retrospectively screening a set of serum specimens from the region in Ghana believed to have harbored the MPXV-infected rodents involved in the 2003 United States outbreak.

Evidence of Mpox Virus Infection Among Persons Without Characteristic Lesions or Rash Presenting for First Dose of JYNNEOS Vaccine-District of Columbia, August 2022.

We assessed mpox virus prevalence in blood, pharyngeal, and rectal specimens among persons without characteristic rash presenting for JYNNEOS vaccine. Our data indicate that the utility of risk-based screening for mpox in persons without skin lesions or rash via pharyngeal swabs, rectal swabs, and/or blood is likely limited.

An Analysis of Outcomes and Management Strategies for Patients With Cholecystostomy Tubes.

BACKGROUND: Percutaneous cholecystostomy tube (PCT) drainage is an effective management strategy for acute cholecystitis in patients medically unfit for surgery. However, little is known about the fate of patients managed by PCT. We conducted this study to determine tube management outcomes for patients with acute cholecystitis managed by PCT. METHODS: The electronic record was queried to identify patients with acute cholecystitis managed by PCT from 2012-2020. Patients were divided into three groups for analysis: 1) ultimately managed by cholecystectomy, 2) eventual confirmation of distal flow of bile from the gallbladder and tube removal, and 3) tubes left in place without further management. RESULTS: A total of 179 patients with acute cholecystitis treated by PCT were included. Sixty-six patients never fully recovered from the medical insult associated with their diagnosis of acute cholecystitis and had their tubes left in situ. Sixty-four of these 66 patients (97%) died during follow-up. The remaining 113 patients recovered from their illness and presented to clinic for evaluation for tube removal and/or cholecystectomy. When distal biliary flow was confirmed, tube removal was favored (n = 70). When cystic duct outflow occlusion persisted, cholecystectomy was planned for patients who became acceptable surgical candidates (n = 43). For patients managed by cholecystectomy, 8 were approached open and 35 laparoscopically, with 12 of 35 (34.3%) converted to open and 23 (65.7%) completed laparoscopically. CONCLUSION: Our study favors PCT removal for patients who recover from their acute illness when distal bile flow from the gallbladder is confirmed. We reserve cholecystectomy for patients who recover from their illness and demonstrate persistent cystic duct outflow obstruction.

Serological responses to the MVA-based JYNNEOS monkeypox vaccine in a cohort of participants from the Democratic Republic of Congo.

The current worldwide monkepox outbreak has reaffirmed the continued threat monkeypox virus (MPXV) poses to public health. JYNNEOS, a Modified Vaccinia Ankara (MVA)-based live, non-replicating vaccine, was recently approved for monkeypox prevention for adults at high risk of MPXV infection in the United States. Although the safety and immunogenicity of JYNNEOS have been examined previously, the clinical cohorts studied largely derive from regions where MPXV does not typically circulate. In this study, we assess the quality and longevity of serological responses to two doses of JYNNEOS vaccine in a large cohort of healthcare workers from the Democratic Republic of Congo (DRC). We show that JYNNEOS elicits a strong orthopoxvirus (OPXV)-specific antibody response in participants that peaks around day 42, or 2 weeks after the second vaccine dose. Participants with no prior history of smallpox vaccination or exposure have lower baseline antibody levels, but experience a similar fold-rise in antibody titers by day 42 as those with a prior history of vaccination. Both previously naïve and vaccinated participants generate vaccinia virus and MPXV-neutralizing antibody in response to JYNNEOS vaccination. Finally, even though total OPXV-specific IgG titers and neutralizing antibody titers declined from their peak and returned close to baseline levels by the 2-year mark, most participants remain IgG seropositive at the 2-year timepoint. Taken together, our data demonstrates that JYNNEOS vaccination triggers potent OPXV neutralizing antibody responses in a cohort of healthcare workers in DRC, a monkeypox-endemic region. MPXV vaccination with JYNNEOS may help ameliorate the disease and economic burden associated with monkeypox and combat potential outbreaks in areas with active virus circulation.

Overcoming Preclinical Safety Obstacles to Discover (S)-N-((1,2,3,5,6,7-Hexahydro-s-indacen-4-yl)carbamoyl)-6-(methylamino)-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-sulfonamide (GDC-2394): A Potent and Selective NLRP3 Inhibitor.

Inappropriate activation of the NLRP3 inflammasome has been implicated in multiple inflammatory and autoimmune diseases. Herein, we aimed to develop novel NLRP3 inhibitors that could minimize the risk of drug-induced liver injury. Lipophilic ligand efficiency was used as a guiding metric to identify a series of 6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazinesulfonylureas. A leading compound from this series was advanced into safety studies in cynomolgus monkeys, and renal toxicity, due to compound precipitation, was observed. To overcome this obstacle, we focused on improving the solubility of our compounds, specifically by introducing basic amine substituents into the scaffold. This led to the identification of GDC-2394, a potent and selective NLRP3 inhibitor, with an in vitro and in vivo safety profile suitable for advancement into human clinical trials.