Modulation of AAV9 Galactose Binding Yields Novel Gene Therapy Vectors and Predicts Cross-Species Differences in Glycan Avidity.

Effective use of adeno-associated viruses (AAVs) for clinical gene therapy is limited by their propensity to accumulate in and transduce the liver. This natural liver tropism is associated with severe adverse events at the high doses that can be necessary for achieving therapeutic transgene expression in extrahepatic tissues. To improve the safety and cost of AAV gene therapy, capsid engineering efforts are underway to redirect in vivo AAV biodistribution away from the liver toward disease-relevant peripheral organs such as the heart. Building on previous work, we generated a series of AAV libraries containing variations at three residues (Y446, N470, and W503) of the galactose-binding pocket of the AAV9 VP1 protein. Screening of this library in mice identified the XRH family of variants (Y446X, N470R, and W503H), the strongest of which, HRH, exhibited a 6-fold reduction in liver RNA expression and a 10-fold increase in cardiac RNA expression compared with wild-type AAV9 in the mouse. Screening of our library in a nonhuman primate (NHP) revealed reduced performance of AAV9 and two closely related vectors in the NHP liver compared with the mouse liver. Measurement of the galactose-binding capacity of our library further identified those same three vectors as the only strong galactose binders, suggesting an altered galactose presentation between the mouse and NHP liver. N-glycan profiling of these tissues revealed a 9% decrease in exposed galactose in the NHP liver compared with the mouse liver. In this work, we identified a novel family of AAV variants with desirable biodistribution properties that may be suitable for targeting extrahepatic tissues such as the heart. These data also provide important insights regarding species- and tissue-specific differences in glycan presentation that may have implications for the development and translation of AAV gene therapies.

High activity of an affinity-matured ACE2 decoy against Omicron SARS-CoV-2 and pre-emergent coronaviruses.

The viral genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), particularly its cell-binding spike protein gene, has undergone rapid evolution during the coronavirus disease 2019 (COVID-19) pandemic. Variants including Omicron BA.1 and Omicron BA.2 now seriously threaten the efficacy of therapeutic monoclonal antibodies and vaccines that target the spike protein. Viral evolution over a much longer timescale has generated a wide range of genetically distinct sarbecoviruses in animal populations, including the pandemic viruses SARS-CoV-2 and SARS-CoV-1. The genetic diversity and widespread zoonotic potential of this group complicates current attempts to develop drugs in preparation for the next sarbecovirus pandemic. Receptor-based decoy inhibitors can target a wide range of viral strains with a common receptor and may have intrinsic resistance to escape mutant generation and antigenic drift. We previously generated an affinity-matured decoy inhibitor based on the receptor target of the SARS-CoV-2 spike protein, angiotensin-converting enzyme 2 (ACE2), and deployed it in a recombinant adeno-associated virus vector (rAAV) for intranasal delivery and passive prophylaxis against COVID-19. Here, we demonstrate the exceptional binding and neutralizing potency of this ACE2 decoy against SARS-CoV-2 variants including Omicron BA.1 and Omicron BA.2. Tight decoy binding tracks with human ACE2 binding of viral spike receptor-binding domains across diverse clades of coronaviruses. Furthermore, in a coronavirus that cannot bind human ACE2, a variant that acquired human ACE2 binding was bound by the decoy with nanomolar affinity. Considering these results, we discuss a strategy of decoy-based treatment and passive protection to mitigate the ongoing COVID-19 pandemic and future airway virus threats.

Intranasal gene therapy to prevent infection by SARS-CoV-2 variants.

SARS-CoV-2 variants have emerged with enhanced pathogenicity and transmissibility, and escape from pre-existing immunity, suggesting first-generation vaccines and monoclonal antibodies may now be less effective. Here we present an approach for preventing clinical sequelae and the spread of SARS-CoV-2 variants. First, we affinity matured an angiotensin-converting enzyme 2 (ACE2) decoy protein, achieving 1000-fold binding improvements that extend across a wide range of SARS-CoV-2 variants and distantly related, ACE2-dependent coronaviruses. Next, we demonstrated the expression of this decoy in proximal airway when delivered via intranasal administration of an AAV vector. This intervention significantly diminished clinical and pathologic consequences of SARS-CoV-2 challenge in a mouse model and achieved therapeutic levels of decoy expression at the surface of proximal airways when delivered intranasally to nonhuman primates. Importantly, this long-lasting, passive protection approach is applicable in vulnerable populations such as the elderly and immune-compromised that do not respond well to traditional vaccination. This approach could be useful in combating COVID-19 surges caused by SARS-CoV-2 variants and should be considered as a countermeasure to future pandemics caused by one of the many pre-emergent, ACE2-dependent CoVs that are poised for zoonosis.

Prevalence of gastrointestinal pathogenic bacteria in patients with diarrhoea attending Groote Schuur Hospital, Cape Town, South Africa.

BACKGROUND: Diarrhoea due to gastrointestinal infections is a significant problem facing the South African (SA) healthcare system. Infections can be acquired both from the community and from the hospital environment itself, the latter acting as a reservoir for potential pathogenic bacteria. OBJECTIVES: To examine the prevalence of a panel of potential diarrhoea-causing bacteria in patients attending a tertiary healthcare facility in Cape Town, SA. METHODS: Polymerase chain reaction (PCR) primers specific for Clostridium difficile, Shigella spp., Salmonella spp., Klebsiella oxytoca, enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC/EHEC), Staphylococcus aureus, enterotoxigenic Bacteroides fragilis and Campylobacter spp. were used to screen total bacterial genomic DNA extracted from stool samples provided by 156 patients with diarrhoea attending Groote Schuur Hospital, Cape Town, SA. RESULTS: C. difficile was the most frequently detected pathogen (16% of cases) in the 21-87-year-old patient range, but was not present in samples from the 16-20-year-old range. K. oxytoca (6%), EPEC/EHEC strains (9%) and S. aureus (6%) were also detected. The remaining pathogens were present at low frequencies (0-2.9%), and the occurrence of mixed infections was 5%. The majority of non-C. difficile-related diarrhoeas were community acquired. CONCLUSION: C. difficile was the main cause of infectious diarrhoea in the sampled patients, while K. oxytoca and EPEC/EHEC strains were present as relatively minor but potentially significant pathogens.

The occurrence of antibiotic resistance genes in drug resistant Bacteroides fragilis isolates from Groote Schuur Hospital, South Africa.

Bacteroides fragilis, an anaerobic gut commensal and opportunistic pathogen, is a leading cause of anaerobic abscesses and bacteraemias. Treatment of infections is complicated by the emergence of resistance to several of the antibiotics used in the clinical setting. Genetic analysis of 23 B. fragilis isolates found that none of the metronidazole resistant strains carried the nimA-J genes, and no cfxA or ermF genes were detected. All of the tetracycline resistant isolates contained the tetQ gene and were sensitive to tigecycline. The cfiA gene was found in 3 of the strains, one of which was imipenem resistant and contained an upstream IS4351 insertion sequence. Another resistant strain had a unique G to A substitution in the promoter region of the cfiA gene, while the third was imipenem sensitive. Thirty percent of the isolates contained at least one plasmid, however, tetQ gene was located on the chromosome and not on any of the plasmids.

Two multidrug-resistant clinical isolates of Bacteroides fragilis carry a novel metronidazole resistance nim gene (nimJ).

Two multidrug-resistant Bacteroides fragilis clinical isolates contain and express a novel nim gene, nimJ, that is not recognized by the "universal" nim primers and can confer increased resistance to metronidazole when introduced into a susceptible strain on a multicopy plasmid. HMW615, an appendiceal isolate, contains at least two copies of nimJ on its genome, while HMW616, an isolate from a patient with sepsis, contains one genomic copy of nimJ. B. fragilis NimJ is phylogenetically closer to Prevotella baroniae NimI and Clostridium botulinum NimA than to the other known Bacteroides Nim proteins. The predicted protein structure of NimJ, based on fold recognition analysis, is consistent with the crystal structures derived for known Nim proteins, and specific amino acid residues important for substrate binding in the active site are conserved. This study demonstrates that the "universal" nim primers will not detect all nim genes with the ability to confer metronidazole resistance, but nimJ alone cannot account for the very high metronidazole MICs of these resistant clinical isolates.