Preclinical Immunopharmacologic Assessment of KPL-404, a Novel, Humanized, Non-Depleting Antagonistic Anti-CD40 Monoclonal Antibody.

The CD40/CD40L pathway plays a major role in multiple inflammatory processes involving different immune and stromal cells. Abnormal activation of this pathway has been implicated in pathogenesis of complex autoimmune diseases including systemic lupus erythematosus, rheumatoid arthritis, Graves' disease, and Sjogren's Syndrome. We completed in vitro and in vivo preclinical characterization of KPL-404, a novel humanized anti-CD40 IgG4 monoclonal antibody, to demonstrate its potency, efficacy, and pharmacokinetic profile; safety was also assessed. In vitro, KPL-404 bound recombinant human and cynomolgus monkey CD40 with comparable affinity in the nanomolar range. KPL-404 binding to cell surface CD40 did not induce antibody- or complement-mediated cytotoxicity of CD40-expressing cells. Pharmacological antagonistic activity of KPL-404 was demonstrated in vitro by inhibition of CD40-mediated downstream NF-kB activation. In the in vivo study with cynomolgus monkeys, KPL-404, administered intravenously as a single dose (10 mg/kg) or two monthly doses of 1 or 5 mg/kg, did not elicit observable safety findings, including thrombocytopenia over 8 weeks. KPL-404 engaged CD40 expressed on peripheral B cells for 2 and 4 weeks after a single administration of 5 or 10 mg/kg IV, respectively, without depletion of peripheral B cells. At 5 mg/kg IV, KPL-404 blocked both primary and secondary responses to T-cell dependent antibody responses to test antigens, KLH, and tetanus toxoid. These data illustrated the relationship between KPL-404 serum concentration and pharmacodynamic effects of CD40-targeting in circulation and in lymphoid tissues. These data support clinical development of KPL-404 in autoimmune diseases. SIGNIFICANCE STATEMENT: We aimed to develop a potent and efficacious CD40 antagonist. In vitro and in vivo findings show that KPL-404 blocks the anti-CD40 antibody that potently inhibits primary and secondary antibody responses at pharmacologically relevant concentrations, has a favorable pharmacokinetic profile, and does not deplete B cells by antibody-dependent cellular cytotoxicity or apoptosis ("nondepleting"). These findings support clinical development of KPL-404 as a potential therapeutic in autoimmune diseases.

Modeling the Potential Future Distribution of Anthrax Outbreaks under Multiple Climate Change Scenarios for Kenya.

The climate is changing, and such changes are projected to cause global increase in the prevalence and geographic ranges of infectious diseases such as anthrax. There is limited knowledge in the tropics with regards to expected impacts of climate change on anthrax outbreaks. We determined the future distribution of anthrax in Kenya with representative concentration pathways (RCP) 4.5 and 8.5 for year 2055. Ecological niche modelling (ENM) of boosted regression trees (BRT) was applied in predicting the potential geographic distribution of anthrax for current and future climatic conditions. The models were fitted with presence-only anthrax occurrences (n = 178) from historical archives (2011-2017), sporadic outbreak surveys (2017-2018), and active surveillance (2019-2020). The selected environmental variables in order of importance included rainfall of wettest month, mean precipitation (February, October, December, July), annual temperature range, temperature seasonality, length of longest dry season, potential evapotranspiration and slope. We found a general anthrax risk areal expansion i.e., current, 36,131 km2, RCP 4.5, 40,012 km2, and RCP 8.5, 39,835 km2. The distribution exhibited a northward shift from current to future. This prediction of the potential anthrax distribution under changing climates can inform anticipatory measures to mitigate future anthrax risk.

Isolation and characterization of a novel Rickettsia species (Rickettsia asembonensis sp. nov.) obtained from cat fleas (Ctenocephalides felis).

A novel rickettsial agent, 'Candidatus Rickettsia asembonensis' strain NMRCiiT, was isolated from cat fleas, Ctenocephalides felis, from Kenya. Genotypic characterization of the new isolate based on sequence analysis of five rickettsial genes, rrs, gltA, ompA, ompB and sca4, indicated that this isolate clustered with Rickettsia felis URRWXCal2. The degree of nucleotide similarity demonstrated that isolate NMRCiiT belongs within the genus Rickettsia and fulfils the criteria for classification as a representative of a novel species. The name Rickettsia asembonensis sp. nov. is proposed, with NMRCiiT (=DSM 100172T=CDC CRIRC RAS001T=ATCC VR-1827T) as the type strain.

Uptake and Effectiveness of a Trivalent Inactivated Influenza Vaccine in Children in Urban and Rural Kenya, 2010 to 2012.

BACKGROUND: In Africa, recent surveillance has demonstrated a high burden of influenza, but influenza vaccine is rarely used. In Kenya, a country with a tropical climate, influenza has been shown to circulate year-round, like in other tropical countries. METHODS: During 3 months in 2010 and 2011 and 2 months in 2012, the Kenya Medical Research Institute/Centers for Disease Control and Prevention-Kenya offered free injectable trivalent inactivated influenza vaccine to children 6 months to 10 years old in 2 resource-poor communities in Kenya-Kibera and Lwak (total population ~50,000). We conducted a case-control study to evaluate vaccine effectiveness (VE) in preventing laboratory-confirmed influenza associated with influenza-like illness and acute lower respiratory illness. RESULTS: Of the approximately 18,000 eligible children, 41%, 48% and 51% received at least 1 vaccine in 2010, 2011 and 2012, respectively; 30%, 36% and 38% were fully vaccinated. VE among fully vaccinated children was 57% [95% confidence interval (CI): 29% to 74%] during a 6-month follow-up period, 39% (95% CI: 17% to 56%) during a 9-month follow-up period and 48% (95% CI: 32% to 61%) during a 12-month follow-up period. For the 12-month follow-up period, VE was statistically significant in children <5 years and in children 5 to <10 years old (50% and 46%, respectively). CONCLUSIONS: In Kenya, parents of nearly half of the eligible children <10 years old chose to get their children vaccinated with a free influenza vaccine. During a 12-month follow-up period, the vaccine was moderately effective in preventing medically attended influenza-associated respiratory illness.

Results from the first six years of national sentinel surveillance for influenza in Kenya, July 2007-June 2013.

BACKGROUND: Recent studies have shown that influenza is associated with significant disease burden in many countries in the tropics, but until recently national surveillance for influenza was not conducted in most countries in Africa. METHODS: In 2007, the Kenyan Ministry of Health with technical support from the CDC-Kenya established a national sentinel surveillance system for influenza. At 11 hospitals, for every hospitalized patient with severe acute respiratory illness (SARI), and for the first three outpatients with influenza-like illness (ILI) per day, we collected both nasopharyngeal and oropharyngeal swabs. Beginning in 2008, we conducted in-hospital follow-up for SARI patients to determine outcome. Specimens were tested by real time RT-PCR for influenza A and B. Influenza A-positive specimens were subtyped for H1, H3, H5, and (beginning in May 2009) A(H1N1)pdm09. RESULTS: From July 1, 2007 through June 30, 2013, we collected specimens from 24,762 SARI and 14,013 ILI patients. For SARI and ILI case-patients, the median ages were 12 months and 16 months, respectively, and 44% and 47% were female. In all, 2,378 (9.6%) SARI cases and 2,041 (14.6%) ILI cases were positive for influenza viruses. Most influenza-associated SARI cases (58.6%) were in children <2 years old. Of all influenza-positive specimens, 78% were influenza A, 21% were influenza B, and 1% were influenza A/B coinfections. Influenza circulated in every month. In four of the six years influenza activity peaked during July-November. Of 9,419 SARI patients, 2.7% died; the median length of hospitalization was 4 days. CONCLUSIONS: During six years of surveillance in Kenya, influenza was associated with nearly 10 percent of hospitalized SARI cases and one-sixth of outpatient ILI cases. Most influenza-associated SARI and ILI cases were in children <2 years old; interventions to reduce the burden of influenza, such as vaccine, could consider young children as a priority group.

Multi-site analytical validation of an assay to detect anti-JCV antibodies in human serum and plasma.

BACKGROUND: JC virus (JCV) infection is a prerequisite for development of progressive multifocal leukoencephalopathy (PML). We previously described the development of a novel, two-step enzyme-linked immunosorbent assay (ELISA) that detects anti-JCV antibodies in human serum or plasma, and the potential clinical utility of anti-JCV antibody status for PML risk stratification. OBJECTIVES: To validate the anti-JCV antibody ELISA at multiple clinical laboratories in order to demonstrate the robustness of the method. STUDY DESIGN: Analytical validation of the ELISA was performed at four laboratories by evaluation of intra- and inter-assay precision, analytical specificity and sensitivity, matrix interference, robustness, sample and reagent stability. RESULTS: Analytical validation demonstrated that the assay is sensitive, specific, and precise. The assay sensitivity was estimated at 1.7ng/mL using a humanized anti-JCV monoclonal antibody control. The sensitivity to detect JCV infection was estimated to be 97.5%. The specificity of the assay to discriminate JCV-specific antibodies from antibodies directed to BK virus, a related polyomavirus, was also demonstrated. The inter- and intra-assay precision was ≤6.0% and 9.8% for the screening step and 2.6% and 11.3% for the confirmation step. Results obtained for plasma and serum were highly congruent, and assay robustness was demonstrated by the highly concordant results generated by four laboratories testing a common panel of 100 blinded samples. CONCLUSIONS: The novel, two-step ELISA to detect anti-JCV antibodies in human serum and plasma is robust, and assay performance is consistent and reproducible in multiple laboratories, making it suitable to support testing for global clinical studies.

Pathogenesis of coxsackievirus-B5 acquired from intra-renal porcine islet cell xenografts in diabetic mice.

BACKGROUND: We previously demonstrated the ability of a human isolate of coxsackievirus-B5 (CVB5) to infect productively adult porcine islet cells (PICs) in vitro. PICs infected with CVB5 remain viable, and upon transplantation reversed diabetes in C56BL/6 mice for up to 5 days. METHODS: In the present work, we expanded this graft-to-host xenozoonosis model by examining the long-term functionality of CVB5-infected PIC xenografts in immunosuppressed mice. And, we characterized the pathogenesis of CVB5 infection in mice resulting from directional transmission of the virus from PIC xenografts to surrounding tissues in a mouse model for immunosuppressed human PIC xenograft recipients. RESULTS: Both acutely (12 h) and chronically (72 h) infected PIC xenografts functioned in vivo to reverse diabetes in mice. The efficacy of both infected and un-infected PICs was transient beyond 5 days post-inoculation and the long-term functionality of the grafts was compromised by host-to-graft rejection. CVB5-infected PIC xenografts transmitted infectious virus to immunosuppressed recipient mice resulting in extensive histopathologic changes. The virus replicated in the heart, liver, spleen, kidney, pancreas, brain and skeletal muscle in higher levels in severe-combined immunodeficient (SCID) mice that were directly inoculated with virus when compared to controls. In addition, infectious virus was recovered for up to 22 days after inoculation in SCID mice whereas it was only detected up to Day 4 PI in non-SCID mice. CONCLUSIONS: Immunosuppressed PIC xenograft recipients may be more susceptible to infection with CVB5 which could target the xenograft leading to disseminated infection in the host.