Modeling antibody drug conjugate potential using a granzyme B antibody fusion protein.

BACKGROUND: Antibody drug conjugates (ADCs) constitute a promising class of targeted anti-tumor therapeutics that harness the selectivity of monoclonal antibodies with the potency of cytotoxic drugs. ADC development is best suited to initially screening antibody candidates for desired properties that potentiate target cell cytotoxicity. However, validating and producing an optimally designed ADC requires expertise and resources not readily available to certain laboratories. RESULTS: In this study, we propose a novel approach to help streamline the identification of potential ADC candidates by utilizing a granzyme B (GrB)-based antibody fusion protein (AFP) for preliminary screening. GrB is a non-immunogenic serine protease expressed by immune effector cells such as CD8 + T cells that induces apoptotic activity and can be leveraged for targeted cell killing. CONCLUSIONS: Our innovative model allows critical antibody parameters (including target cell binding, internalization, and cytotoxic potential) to be more reliably evaluated in vitro through the creation of an ADC surrogate. Successful incorporation of this AFP could also significantly expand and enhance ADC development pre-clinically, ultimately leading to the accelerated translation of ADC therapies for patients.

Serratia marcescens enzyme SME-2 isolated from sputum in New Zealand.

Introduction: The Serratia marcescens enzymes (SMEs) are chromosomally encoded Ambler Class A carbapenem-hydrolysing ß-lactamases, which distinctively express resistance to carbapenems while remaining susceptible to extended-spectrum cephalosporins. Global reports of SMEs are infrequent. Here we describe the isolation of an SME-2-producing S. marcescens from the sputum of a patient who was hospitalized at Christchurch Hospital, New Zealand. Methods: An immunosuppressed asthmatic patient who presented with shortness of breath and hypoxia grew S. marcescens from a sputum culture. Antimicrobial susceptibilities were determined by Phoenix, with MICs of meropenem and imipenem determined by Liofilchem® MIC gradient strips and interpreted according to EUCAST breakpoints. Investigation for carbapenemase was performed using Carba NP, modified CIM (mCIM) and GeneXpert® Carba-R. WGS was performed using the Illumina DNA Prep library kit and sequenced using MiSeq. Results: The isolate showed an unusual susceptibility profile, including high-level resistance to meropenem and imipenem, while remaining susceptible to extended-spectrum cephalosporins. The Carba NP and mCIM were positive and WGS demonstrated the presence of a blaSME-2 gene located on the chromosome within the SmarGI1-1 genomic island. In addition, a blaSRT-like class C ß-lactamase, aac(6')-Ic aminoglycoside-modifying enzyme and various multidrug efflux mechanisms were found. Phylogenetic core-genome analysis indicated no matching genome with RefSeq database strains. Conclusions: S. marcescens is an opportunistic pathogen of concern, harbouring a variety of intrinsic resistance mechanisms, including the potential for stable AmpC hyperproduction. Globally, SME-type carbapenemases have been infrequently reported; however, isolates carrying this mechanism could have limited treated options, having implications for patient management. To the best of our knowledge this is the first report of SME in New Zealand.

Listeria-based vaccination against the pericyte antigen RGS5 elicits anti-vascular effects and colon cancer protection.

Colorectal cancer (CRC) remains a leading cause of cancer-related mortality despite efforts to improve standard interventions. As CRC patients can benefit from immunotherapeutic strategies that incite effector T cell action, cancer vaccines represent a safe and promising therapeutic approach to elicit protective and durable immune responses against components of the tumor microenvironment (TME). In this study, we investigate the pre-clinical potential of a Listeria monocytogenes (Lm)-based vaccine targeting the CRC-associated vasculature. CRC survival and progression are reliant on functioning blood vessels to effectively mediate various metabolic processes and oxygenate underlying tissues. We, therefore, advance the strategy of initiating immunity in syngeneic mouse models against the endogenous pericyte antigen RGS5, which is a critical mediator of pathological vascularization. Overall, Lm-based vaccination safely induced potent anti-tumor effects that consisted of recruiting functional Type-1-associated T cells into the TME and reducing tumor blood vessel content. This study underscores the promising clinical potential of targeting RGS5 against vascularized tumors like CRC.

Targeting the pericyte antigen DLK1 with an alpha type-1 polarized dendritic cell vaccine results in tumor vascular modulation and protection against colon cancer progression.

Despite the availability of various treatment options, colorectal cancer (CRC) remains a significant contributor to cancer-related mortality. Current standard-of-care interventions, including surgery, chemotherapy, and targeted agents like immune checkpoint blockade and anti-angiogenic therapies, have improved short-term patient outcomes depending on disease stage, but survival rates with metastasis remain low. A promising strategy to enhance the clinical experience with CRC involves the use of dendritic cell (DC) vaccines that incite immunity against tumor-derived blood vessels, which are necessary for CRC growth and progression. In this report, we target tumor-derived pericytes expressing DLK1 with a clinically-relevant alpha type-1 polarized DC vaccine (αDC1) in a syngeneic mouse model of colorectal cancer. Our pre-clinical data demonstrate the αDC1 vaccine's ability to induce anti-tumor effects by facilitating cytotoxic T lymphocyte activity and ablating the tumor vasculature. This work, overall, provides a foundation to further interrogate immune-mediated mechanisms of protection in order to help devise efficacious αDC1-based strategies for patients with CRC.

Anterior and posterior imaging with hyperparallel OCT.

Hyperparallel OCT (HP-OCT) is a parallel spectral domain imaging technology particularly well-suited to the anterior segment. It uses a 2-dimensional grid of 1008 beams to simultaneously image across a wide area of the eye. In this paper we demonstrate that sparsely sampled volumes captured at 300 Hz can be registered without the need for active eye tracking to produce 3-dimensional (3D) volumes free from motion artefacts. The anterior volume provides complete 3D biometric information, including lens position, curvature, epithelial thickness, tilt, and axial length. We further demonstrate that, with the change of a detachable lens, we can capture high resolution anterior volumes and importantly, posterior volume images for preoperative assessment of the posterior segment. Advantageously, the retinal volumes have the same 11.2 mm Nyquist range as the anterior imaging mode.

A pilot study using unique targeted testing of the urogenital microbiome has potential as a predictive test during IVF for implantation outcome.

PURPOSE: This pilot study aimed to develop a methodology characterising the urogenital microbiome as a predictive test in the IVF workup. METHODS: Using unique custom qPCRs, we tested for the presence of specific microbial species from vaginal samples and First Catch Urines from the male. The test panel included a range of potential urogenital pathogens, STIs, 'favourable bacteria' (Lactobacillus spp.) and 'unfavourable bacteria' (anaerobes) reported to influence implantation rates. We tested couples attending Fertility Associates, Christchurch, New Zealand for their first round of IVF. RESULTS: We found that some microbial species affected implantation. The qPCR result was interpreted qualitatively using the Z proportionality test. Samples from women at the time of Embryo Transfer who did not achieve implantation had significantly higher percent of samples that were positive for Prevotella bivia and Staphylococcus aureus compared to women who did achieve implantation. DISCUSSION: The results provide evidence that most other microbial species chosen for testing had little functional effect on implantation rates. The addition of further microbial targets (yet to be determined) could be combined in this predictive test for vaginal preparedness on the day of embryo transfer. This methodology has a substantial advantage of being affordable and easily performed in any routine molecular laboratory. This methodology is most suitable as a foundation on which to develop a timely test of microbiome profiling. Using the indicators detected to have a significant influence, these results can be extrapolated. CONCLUSION: Using a rapid antigen test, a woman can self-sample prior to embryo transfer and obtain an indication of microbial species present which could influence implantation outcome.

Retinal magnification factors at the fixation locus derived from schematic eyes with four individualized surfaces.

Retinal magnification factors (RMFs) allow the conversion of angles to lengths in retinal images. In this work, we propose paraxial and non-paraxial RMF calculation methods that incorporate the individual topography and separation of the anterior and posterior surfaces of the cornea and crystalline lens, assuming homogeneous ocular media. Across 34 eyes, the two RMF methods differ by 0.1% on average, due to surface tilt, decenter, and lack of rotational symmetry in the non-paraxial modeling, which results in up to 2.2% RMF variation with retinal meridian. Differences with widely used individualized RMF calculation methods are smallest for eyes with ∼24 mm axial length, and as large as 7.5% in a 29.7 mm long eye (15D myope). To better model the capture of retinal images, we propose the tracing of chief rays, instead of the scaling of posterior nodal or principal distances often used in RMF definitions. We also report that RMF scale change is approximately proportional to both refractive error and axial separation between the ophthalmoscope's exit pupil and the eye's entrance pupil, resulting in RMF changes as large as 13% for a 1cm displacement in a 15D myopic eye. Our biometry data shows weak correlation and statistical significance between surface radii and refractive error, as well as axial length, whether considering all eyes in the study, or just the high myopes, defined as those with refractive error sphere equivalent ≤ -4D. In contrast, vitreous thicknesses show a strong correlation (r ≤ -0.92) and significance (p ≤ 10-13) with refractive error when considering all eyes or just high myopes (r ≤ -0.95; p ≤ 10-5). We also found that potential RMF change with depth of cycloplegia and/or residual accommodation is smaller than 0.2%. Finally, we propose the reporting of individual ocular biometry data and a detailed RMF calculation method description in scientific publications to facilitate the comparison of retinal imaging biomarker data across studies.

Clinical, Laboratory, and Molecular Epidemiology of an Outbreak of Aseptic Meningitis Due to a Triple-Recombinant Echovirus in Ashburton, New Zealand.

Here, we describe a small enterovirus outbreak including nine cases of aseptic meningitis in a New Zealand hospital in 2017. Most patients had a lymphocytic predominance in the CSF, their length of stay was short, and there were no paediatric cases or ICU admissions. VP1 genotyping revealed that the outbreak was caused by an echovirus E30 strain closely related to strains reported from the US, UK, Brazil, and Denmark. They all form a separate cluster within lineage "h", which leads to the proposal of establishing a new lineage tentatively named "j" for this group of echovirus E30 strains. However, whole genome sequencing and reference mapping to echovirus E30 sequences showed very poor mapping of reads to the 3' half of the genome. Further bioinformatic analysis indicated that the causative agent of this outbreak might be a mosaic triple-recombinant enterovirus composed of echovirus E6, echovirus E11, and echovirus E30 genome segments.

Extensive epigenetic modification with large-scale chromosomal and plasmid recombination characterise the Legionella longbeachae serogroup 1 genome.

Legionella longbeachae is an environmental bacterium that is the most clinically significant Legionella species in New Zealand (NZ), causing around two-thirds of all notified cases of Legionnaires' disease. Here we report the sequencing and analysis of the geo-temporal genetic diversity of 54 L. longbeachae serogroup 1 (sg1) clinical isolates, derived from cases from around NZ over a 22-year period, including one complete genome and its associated methylome. The 54 sg1 isolates belonged to two main clades that last shared a common ancestor between 95 BCE and 1694 CE. There was diversity at the genome-structural level, with large-scale arrangements occurring in some regions of the chromosome and evidence of extensive chromosomal and plasmid recombination. This includes the presence of plasmids derived from recombination and horizontal gene transfer between various Legionella species, indicating there has been both intra- and inter-species gene flow. However, because similar plasmids were found among isolates within each clade, plasmid recombination events may pre-empt the emergence of new L. longbeachae strains. Our complete NZ reference genome consisted of a 4.1 Mb chromosome and a 108 kb plasmid. The genome was highly methylated with two known epigenetic modifications, m4C and m6A, occurring in particular sequence motifs within the genome.

Disrupting cancer angiogenesis and immune checkpoint networks for improved tumor immunity.

Immune checkpoint inhibitors (ICIs) have advanced the field of cancer immunotherapy in patients by sustaining effector immune cell activity within the tumor microenvironment. However, the approach in general is still faced with issues related to ICI response duration/resistance, treatment eligibility, and safety, which indicates a need for further refinements. As immune checkpoint upregulation is inextricably linked to cancer-induced angiogenesis, newer clinical efforts have demonstrated the feasibility of disrupting both tumor-promoting networks to mediate enhanced immune-driven protection. This review focuses on such key evidence stipulating the necessity of co-applying ICI and anti-angiogenic strategies in cancer patients, with particular interest in highlighting newer engineered antibody approaches that may provide theoretically superior multi-pronged and safe therapeutic combinations.

Kingella kingae Spondylodiscitis: Treatment Failure With Flucloxacillin.

Kingella kingae infections generally respond well to most beta-lactam antibiotics. We investigated an antibiotic treatment failure in a 3-year-old with K. kingae L3-4 spondylodiscitis. Her disease progressed even after 19 days of high-dose intravenous flucloxacillin. The clinical isolate did not produce a beta-lactamase and despite phenotypic testing and whole-genome sequencing, the mechanism of flucloxacillin resistance remains unknown.

Markers for discriminating Campylobacter concisus genomospecies using MALDI-TOF analysis.

Strains identified as Campylobacter concisus may belong to one of at least two biochemically indistinguishable, but genomically distinct, groups referred to as "genomospecies" that may differ in their pathogenic and zoonotic potential. Reliable, affordable and available identification methods are required to improve understanding of their significance in human illness. We examined the potential for MALDI-TOF MS, increasingly used in routine laboratories, for this task. Nineteen well-characterised strains were examined using a widely used MALDI-TOF MS commercial system, however only one strain confidently identified using their database. Data mining of the spectra obtained revealed a number of markers that could be used to help discriminate these genomospecies. We conclude that careful application of MALDI-TOF analysis could be useful to determine the role and significance of diverse C. concisus genomospecies in human disease.

Whole-genome sequencing and ad hoc shared genome analysis of Staphylococcus aureus isolates from a New Zealand primary school.

Epidemiological studies of communicable diseases increasingly use large whole-genome sequencing (WGS) datasets to explore the transmission of pathogens. It is important to obtain an initial overview of datasets and identify closely related isolates, but this can be challenging with large numbers of isolates and imperfect sequencing. We used an ad hoc whole-genome multi locus sequence typing method to summarise data from a longitudinal study of Staphylococcus aureus in a primary school in New Zealand. Each pair of isolates was compared and the number of genes where alleles differed between isolates was tallied to produce a matrix of "allelic differences". We plotted histograms of the number of allelic differences between isolates for: all isolate pairs; pairs of isolates from different individuals; and pairs of isolates from the same individual. 340 sequenced isolates were included, and the ad hoc shared genome contained 445 genes. There were between 0 and 420 allelic differences between isolate pairs and the majority of pairs had more than 260 allelic differences. We found many genetically closely related S. aureus isolates from single individuals and a smaller number of closely-related isolates from separate individuals. Multiple S. aureus isolates from the same individual were usually very closely related or identical over the ad hoc shared genome. Siblings carried genetically similar, but not identical isolates. An ad hoc shared genome approach to WGS analysis can accommodate imperfect sequencing of the included isolates, and can provide insights into relationships between isolates in epidemiological studies with large WGS datasets containing diverse isolates.

Dendritic cell vaccine therapy for colorectal cancer.

Colorectal cancer (CRC) remains a leading cause of cancer-related deaths in the United States despite an array of available treatment options. Current standard-of-care interventions for this malignancy include surgical resection, chemotherapy, and targeted therapies depending on the disease stage. Specifically, infusion of anti-vascular endothelial growth factor agents in combination with chemotherapy was an important development in improving the survival of patients with advanced colorectal cancer, while also helping give rise to other forms of anti-angiogenic therapies. Yet, one approach by which tumor angiogenesis may be further disrupted is through the administration of a dendritic cell (DC) vaccine targeting tumor-derived blood vessels, leading to cytotoxic immune responses that decrease tumor growth and synergize with other systemic therapies. Early generations of such vaccines exhibited protection against various forms of cancer in pre-clinical models, but clinical results have historically been disappointing. Sipuleucel-T (Provenge®) was the first, and to-date, only dendritic cell-based therapy to receive FDA approval after significantly increasing overall survival in prostate cancer patients. The unparalleled success of Sipuleucel-T has helped revitalize the clinical development of dendritic cell vaccines, which will be examined in this review. We also highlight the promise of these vaccines to instill anti-angiogenic immunity for individuals with advanced colorectal cancer.

Antibody-drug conjugates: an evolving approach for melanoma treatment.

Melanoma continues to be an aggressive and deadly form of skin cancer while therapeutic options are continuously developing in an effort to provide long-term solutions for patients. Immunotherapeutic strategies incorporating antibody-drug conjugates (ADCs) have seen varied levels of success across tumor types and represent a promising approach for melanoma. This review will explore the successes of FDA-approved ADCs to date compared to the ongoing efforts of melanoma-targeting ADCs. The challenges and opportunities for future therapeutic development are also examined to distinguish how ADCs may better impact individuals with malignancies such as melanoma.

Enhancement of Culture of Legionella longbeachae from Respiratory Samples by Use of Immunomagnetic Separation and Antimicrobial Decontamination.

Legionella longbeachae is the commonest Legionella species identified in patients with community-acquired pneumonia in New Zealand. Isolation of the organism on culture is the gold standard for the diagnosis of Legionnaires disease, but it has poor sensitivity (40%) compared with quantitative PCR (qPCR). We have developed a selective decontamination process using glycine, vancomycin, polymyxin, and cycloheximide (GVPC) with immunomagnetic separation (IMS) for culturing L. longbeachae A polyclonal antibody specific for L. longbeachae was produced from New Zealand White rabbits and coupled to tosyl-activated magnetic beads. Stored L. longbeachae qPCR-positive respiratory samples were retrieved from -80°C storage for testing. One portion of test samples was mixed with GVPC and the antibody bead complex, separated, washed, and cultured on modified Wadowsky and Yee agar (MWY) agar. Another portion was exposed to HCl-KCl acidic buffer (pH 2.2) before incubation on MWY agar. qPCR used probes specific for the ITS (internal transcribed spacer) region of the L. longbeachae genome. Cultures were positive in 10/53 (19%) samples after acid wash and 26/53 (49%) after GVPC-IMS (P = 0.001). Growth of contaminants was rare. The mean qPCR threshold cycle values were lower in culture-positive samples after acid wash than in the culture-negative samples (mean, 29.9 versus 34.8; difference, 4.9; 95% confidence interval [CI], ±2.9; P = 0.001) but not after GVPC-IMS (mean, 33.0 versus 34.7; difference, 1.7; 95% CI, ±2.48; P = 0.16). The sensitivity of culture for L. longbeachae in respiratory specimens may be improved by using GVPC-IMS rather than acid wash for decontamination, but this should be confirmed in a prospective study of fresh specimens.

Complete Genome Sequence of a Legionella longbeachae Serogroup 2 Isolate Derived from a Patient with Legionnaires' Disease.

Legionella longbeachae is the predominant cause of Legionnaires' disease (LD) in New Zealand. Although serogroup 2 (sg2) does not contain the most clinically significant strain, it is an important cause of disease. Here, we report the complete genome sequence of an sg2 isolate from a patient who was hospitalized with LD.

Elizabethkingia miricola bacteraemia in a haemodialysis patient.

We report a case of catheter-associated Elizabethkingia miricola bacteraemia in a haemodialysis patient. The patient was a 73-year-old home haemodialysis patient who presented with a history of recurrent falls and fevers. Blood cultures grew Gram-negative bacilli identified by MALDI-TOF MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry 6903 MSP Library) and 16S rRNA gene sequencing as E. miricola. E. miricola is an emerging human pathogen and is multidrug-resistant, making the choice of antimicrobial therapy challenging. There are only a small number of case reports of human infection worldwide and this is the second reported case of catheter-related bacteraemia. It has also been found in the hospital environment in South Korea and is pathogenic in black-spotted frogs.

The burden of Legionnaires' disease in New Zealand (LegiNZ): a national surveillance study.

BACKGROUND: Legionnaires' disease is under-diagnosed because of inconsistent use of diagnostic tests and uncertainty about whom to test. We assessed the increase in case detection following large-scale introduction of routine PCR testing of respiratory specimens in New Zealand. METHODS: LegiNZ was a national surveillance study done over 1-year in which active case-finding was used to maximise the identification of cases of Legionnaires' disease in hospitals. Respiratory specimens from patients of any age with pneumonia, who could provide an eligible lower respiratory specimen, admitted to one of 20 participating hospitals, covering a catchment area of 96% of New Zealand's population, were routinely tested for legionella by PCR. Additional cases of Legionnaires' disease in hospital were identified through mandatory notification. FINDINGS: Between May 21, 2015, and May 20, 2016, 5622 eligible specimens from 4862 patients were tested by PCR. From these, 197 cases of Legionnaires' disease were detected. An additional 41 cases were identified from notification data, giving 238 cases requiring hospitalisation. The overall incidence of Legionnaires' disease cases in hospital in the study area was 5·4 per 100 000 people per year, and Legionella longbeachae was the predominant cause, found in 150 (63%) of 238 cases. INTERPRETATION: The rate of notified disease during the study period was three-times the average over the preceding 3 years. Active case-finding through systematic PCR testing better clarified the regional epidemiology of Legionnaires' disease and uncovered an otherwise hidden burden of disease. These data inform local Legionnaires' disease testing strategies, allow targeted antibiotic therapy, and help identify outbreaks and effective prevention strategies. The same approach might have similar benefits if applied elsewhere in the world. FUNDING: Health Research Council of New Zealand.