Systemic prime mucosal boost significantly increases protective efficacy of bivalent RSV influenza viral vectored vaccine.

Although licensed vaccines against influenza virus have been successful in reducing pathogen-mediated disease, they have been less effective at preventing viral infection of the airways and current seasonal updates to influenza vaccines do not always successfully accommodate viral drift. Most licensed influenza and recently licensed RSV vaccines are administered via the intramuscular route. Alternative immunisation strategies, such as intranasal vaccinations, and "prime-pull" regimens, may deliver a more sterilising form of protection against respiratory viruses. A bivalent ChAdOx1-based vaccine (ChAdOx1-NP + M1-RSVF) encoding conserved nucleoprotein and matrix 1 proteins from influenza A virus and a modified pre-fusion stabilised RSV A F protein, was designed, developed and tested in preclinical animal models. The aim was to induce broad, cross-protective tissue-resident T cells against heterotypic influenza viruses and neutralising antibodies against RSV in the respiratory mucosa and systemically. When administered via an intramuscular prime-intranasal boost (IM-IN) regimen in mice, superior protection was generated against challenge with either RSV A, Influenza A H3N2 or H1N1. These results support further clinical development of a pan influenza & RSV vaccine administered in a prime-pull regimen.

Optimizing a linear 'Doggybone' DNA vaccine for influenza virus through the incorporation of DNA targeting sequences and neuraminidase antigen.

Influenza virus represents a challenge for traditional vaccine approaches due to its seasonal changes and potential for zoonotic transmission. Nucleic acid vaccines can overcome some of these challenges, especially through the inclusion of multiple antigens to increase the breadth of response. RNA vaccines were an important part of the response to the COVID-19 pandemic, but for future outbreaks DNA vaccines may have some advantages in terms of stability and manufacturing cost that warrant continuing investigation to fully realize their potential. Here, we investigate influenza virus vaccines made using a closed linear DNA platform, Doggybone™ DNA (dbDNA), produced by a rapid and scalable cell-free method. Influenza vaccines have mostly focussed on Haemagglutinin (HA), but the inclusion of Neuraminidase (NA) may provide additional protection. Here, we explored the potential of including NA in a dbDNA vaccine, looking at DNA optimization, mechanism and breadth of protection. We showed that DNA targeting sequences (DTS) improved immune responses against HA but not NA. We explored whether NA vaccine-induced protection against influenza virus infection was cell-mediated, but depletion of CD8 and NK cells made no impact, suggesting it was antibody-mediated. This is reflected in the restriction of protection to homologous strains of influenza virus. Importantly, we saw that including both HA and NA in a single combined vaccine did not dampen the immune response to either one. Overall, we show that linear dbDNA can induce an immune response against NA, which may offer increased protection in instances of HA mismatch where NA remains more conserved.

Reducing cell intrinsic immunity to mRNA vaccine alters adaptive immune responses in mice.

The response to mRNA vaccines needs to be sufficient for immune cell activation and recruitment, but moderate enough to ensure efficacious antigen expression. The choice of the cap structure and use of N1-methylpseudouridine (m1Ψ) instead of uridine, which have been shown to reduce RNA sensing by the cellular innate immune system, has led to improved efficacy of mRNA vaccine platforms. Understanding how RNA modifications influence the cell intrinsic immune response may help in the development of more effective mRNA vaccines. In the current study, we compared mRNA vaccines in mice against influenza virus using three different mRNA formats: uridine-containing mRNA (D1-uRNA), m1Ψ-modified mRNA (D1-modRNA), and D1-modRNA with a cap1 structure (cC1-modRNA). D1-uRNA vaccine induced a significantly different gene expression profile to the modified mRNA vaccines, with an up-regulation of Stat1 and RnaseL, and increased systemic inflammation. This result correlated with significantly reduced antigen-specific antibody responses and reduced protection against influenza virus infection compared with D1-modRNA and cC1-modRNA. Incorporation of m1Ψ alone without cap1 improved antibodies, but both modifications were required for the optimum response. Therefore, the incorporation of m1Ψ and cap1 alters protective immunity from mRNA vaccines by altering the innate immune response to the vaccine material.

Formulation, inflammation, and RNA sensing impact the immunogenicity of self-amplifying RNA vaccines.

To be effective, RNA vaccines require both in situ translation and the induction of an immune response to recruit cells to the site of immunization. These factors can pull in opposite directions with the inflammation reducing expression of the vaccine antigen. We investigated how formulation affects the acute systemic cytokine response to a self-amplifying RNA (saRNA) vaccine. We compared a cationic polymer (pABOL), a lipid emulsion (nanostructured lipid carrier, NLC), and three lipid nanoparticles (LNP). After immunization, we measured serum cytokines and compared the response to induced antibodies against influenza virus. Formulations that induced a greater cytokine response induced a greater antibody response, with a significant correlation between IP-10, MCP-1, KC, and antigen-specific antibody titers. We then investigated how innate immune sensing and signaling impacted the adaptive immune response to vaccination with LNP-formulated saRNA. Mice that lacked MAVS and are unable to signal through RIG-I-like receptors had an altered cytokine response to saRNA vaccination and had significantly greater antibody responses than wild-type mice. This indicates that the inflammation induced by formulated saRNA vaccines is not solely deleterious in the induction of antibody responses and that targeting specific aspects of RNA vaccine sensing might improve the quality of the response.

An unusual case of a diffuse targetoid rash.

Capnocytophaga canis is a commensal bacterium present in the oral cavities of dogs and cats. Human infection with Capnocytophaga spp. can present with a range of symptoms from mild flu-like illness to sepsis and disseminated intravascular coagulation. A case fatality rate of up to 31% has been reported. We present a case of C. canis infection presenting with sepsis, purpura, cellulitis and a targetoid rash. Click here for the corresponding questions to this CME article.

Prevalence and characteristics of post-colonoscopy colorectal cancers in a New Zealand regional centre: a 10-year analysis.

BACKGROUND: Post-colonoscopy colorectal cancers (PCCRC) are cancers that appear following a colonoscopy in which no cancer is diagnosed. The occurrence of PCCRC is thought to be multifactorial, reflecting both endoscopy quality and potential differences in tumour biology between detected colorectal cancers and PCCRC. AIM: To identify the prevalence and characteristics of PCCRC in a New Zealand regional centre over a 10-year period. METHOD: All cases of colorectal cancer (n = 1055) in the Bay of Plenty region between 1 February 2009 and 1 February 2019 were cross-referenced with endoscopy coding records to identify patients who had undergone colonoscopy within the preceding 6-60 months in which cancer was not identified. RESULTS: A total of 46 patients were identified to have PCCRC, giving a prevalence of 4.4%. The majority of these patients were older (80% aged 65 years or over) and female (67%). The mean interval between index colonoscopy and diagnosis of PCCRC was 3.03 years. Most (80%) patients had existent pathology (diverticular disease or colonic polyps) at index colonoscopy, and a significant proportion (43%) developed cancer in the same colonic segment. PCCRC were evenly distributed between the left (50%) and right (50%) colon. The majority of patients (63%) had early-stage cancer. CONCLUSIONS: The prevalence of PCCRC in a New Zealand cohort is consistent with other international reports. Most patients with PCCRC are older, female and have early-stage disease. Of interest, a high proportion of patients developed cancer within a colonic segment with existent pathology, suggesting either missed lesions or incomplete polyp resection.

Rational design of adjuvants for subunit vaccines: The format of cationic adjuvants affects the induction of antigen-specific antibody responses.

A range of cationic delivery systems have been investigated as vaccine adjuvants, though few direct comparisons exist. To investigate the impact of the delivery platform, we prepared four cationic systems (emulsions, liposomes, polymeric nanoparticles and solid lipid nanoparticles) all containing equal concentrations of the cationic lipid dimethyldioctadecylammonium bromide in combination with the Neisseria adhesin A variant 3 subunit antigen. The formulations were physicochemically characterized and their ability to associate with cells and promote antigen processing (based on degradation of DQ-OVA, a substrate for proteases which upon hydrolysis is fluorescent) was compared in vitro and their vaccine efficacy (antigen-specific antibody responses and IFN-γ production) and biodistribution (antigen and adjuvant) were evaluated in vivo. Due to their cationic nature, all delivery systems gave high antigen loading (> 85%) with liposomes, lipid nanoparticles and emulsions being <200 nm, whilst polymeric nanoparticles were larger (~350 nm). In vitro, the particulate systems tended to promote cell uptake and antigen processing, whilst emulsions were less effective. Similarly, whilst the particulate delivery systems induced a depot (of both delivery system and antigen) at the injection site, the cationic emulsions did not. However, out of the systems tested the cationic emulsions induced the highest antibody responses. These results demonstrate that while cationic lipids can have strong adjuvant activity, their formulation platform influences their immunogenicity.

Synthesis of protein conjugates adsorbed on cationic liposomes surface.

The well-known Toll like receptor 9 (TLR9) agonist CpG ODN has shown promising results as vaccine adjuvant in preclinical and clinical studies, however its in vivo stability and potential systemic toxicity remain a concern. In an effort to overcome these issues, different strategies have been explored including conjugation of CpG ODN with proteins or encapsulation/adsorption of CpG ODN into/onto liposomes. Although these methods have resulted in enhanced immunopotency compared to co-administration of free CpG ODN and antigen, we believe that this effect could be further improved. Here, we designed a novel delivery system of CpG ODN based on its conjugation to serve as anchor for liposomes. Thiol-maleimide chemistry was utilised to covalently ligate model protein with the CpG ODN TLR9 agonist. Due to its negative charge, the protein conjugate readily electrostatically bound cationic liposomes composed of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol and dimethyldioctadecylammonium bromide (DDA) in a very high degree. The novel cationic liposomes-protein conjugate complex shared similar vesicle characteristics (size and charge) compared to free liposomes. The conjugation of CpG ODN to protein in conjunction with adsorption on cationic liposomes, could promote co-delivery leading to the induction of immune response at low antigen and CpG ODN doses.•The CpG ODN Toll-like receptor (TLR) 9 agonist was conjugated to protein antigens via thiol-maleimide chemistry.•Due to their negative charge, protein conjugates readily electrostatically bound cationic liposomes composed of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol and dimethyldioctadecylammonium bromide (DDA) resulting to the design of novel cationic liposomes-protein conjugate complexes.•The method is suited for the liposomal delivery of a variety of adjuvant-protein conjugates.

Design of a novel vaccine nanotechnology-based delivery system comprising CpGODN-protein conjugate anchored to liposomes.

Although the well-known Toll like receptor 9 (TLR9) agonist CpGODN has shown promising results as vaccine adjuvant in preclinical and clinical studies, its in vivo stability and potential systemic toxicity remain a concern. In an effort to circumvent these issues, different strategies have been employed to increase its stability, localise action and reduce dosage. These include conjugation of CpGODN with proteins or encapsulation/adsorption of CpGODN into/onto liposomes, and have resulted in enhanced immunopotency compared to co-administration of free CpGODN and antigen. Here, we designed a novel delivery system of CpGODN based on its conjugation to serve as anchor for liposomes. Thiol-maleimide chemistry was utilised to covalently ligate the Group B Streptococcus (GBS) GBS67 protein antigen with the CpGODN TLR9 agonist. This treatment did not alter protein's ability to be recognised by specific antibodies or the CpGODN to function as a TLR9 agonist. Due to its negative charge, the protein conjugate readily electrostatically bound cationic liposomes composed of 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol and dimethyldioctadecylammonium bromide (DDA). The novel cationic liposomes-protein conjugate complex (GBS67-CpGODN+L) shared similar vesicle characteristics (size and charge) compared to free liposomes but exhibited different structure and morphology. Following intramuscular immunisation, GBS67-CpGODN+L formed a vaccine depot at the injection site and induced a remarkable increase of functional immune responses against GBS compared to the simple co-administration of GBS67, CpGODN and liposomes. This work demonstrates that the conjugation of CpGODN to GBS67 in conjunction with adsorption on cationic liposomes, can promote co-delivery leading to the induction of a multifaceted immune response at low antigen and CpGODN doses. Our findings highlight the potential for harnessing the immunostimulatory properties of different adjuvants to develop more effective nanostructure-based vaccine platforms.

Medical image. An unusual case of melaena.

Metastatic melanoma can present with non-cutaneous symptoms even after several years of remission. Although poor prognosis, surgical resection and arterial embolisation can provide effective symptom palliation.

The role of capsule endoscopy in small bowel pathology: a review of 122 cases.

AIM: Small bowel capsule endoscopy (CE) has been introduced in New Zealand (NZ) in all of the tertiary and some secondary centres over the last few years. We describe our experience with CE from a single centre in NZ. METHODS: In this 2-year, retrospective, study of 122 consecutive patients, data was collected on multiple variables from the patient clinical, laboratory, and radiology records. Pillcam of Given Imaging Diagnostic System (Given Imaging Ltd, Yogneam, Israel) was used to image the small bowel. Descriptive statistics were used to analyse the data. RESULTS: Good preparation was noted in 69% of the cases. The most common indication for referral was obscure GI bleeding (70%). The overall diagnostic yield for relevant findings was 52%, with angioectasia as the most common specific finding (37%). The diagnostic yield in those with overt bleeds improved with inpatient status (74%). Incomplete examinations were noted in 12% and were significantly more common in the male gender. Preliminary imaging (barium, CT/MR) was noted to have a lower diagnostic yield. Enteroscopies were considered in 25% of the patients post CE procedure. CONCLUSION: Apart from a lower diagnostic yield in patients with overt bleeds, our data is consistent with that reported in literature and support the role of CE as the minimally invasive gold standard investigation for small bowel imaging.

Shewanella algae causing lower limb soft tissue infection in New Zealand.

A 59-year-old man presented to Tauranga Hospital (Tauranga, New Zealand) with lower limb soft tissue infection growing Shewanella algae isolated from blood and skin after fishing in seawater. This is the first published report of this marine organism causing infection in New Zealand.

Developing a predictive understanding of landscape importance to the Punan-Pelancau of East Kalimantan, Borneo.

In order for local community views to be incorporated into new development initiatives, their perceptions need to be clearly understood and documented in a format that is readily accessible to planners and developers. The current study sought to develop a predictive understanding of how the Punan Pelancau community, living in a forested landscape in East Kalimantan, assigns importance to its surrounding landscapes and to present these perceptions in the form of maps. The approach entailed the iterative use of a combination of participatory community evaluation methods and more formal modeling and geographic information system techniques. Results suggest that landscape importance is largely dictated by potential benefits, such as inputs to production, health, and houses. Neither land types nor distance were good predictors of landscape importance. The grid-cell method, developed as part of the study, appears to offer a simple technique to capture and present the knowledge of local communities, even where their relationship to the land is highly complex, as was the case for this particular community.

Artificial nutrition support in intestinal failure: principles and practice of parenteral feeding.

Patients with intestinal failure are at risk for malnutrition and its associated adverse consequences. In many of these patients it is not possible to feed via the gastrointestinal tract, and nutrients must be provided directly into the bloodstream. For some patients with irreversible intestinal failure, this is a lifelong requirement. Parenteral nutrient solutions may be tailored specifically to individual requirements and are usually administered directly into a central vein using an indwelling catheter. Serious complications related to both the indwelling catheter and metabolic consequences of the nutritional support may occur. A team approach to the provision and monitoring of parenteral nutrition in intestinal failure produces the best results.