Vectored immunoprophylaxis and treatment of SARS-CoV-2 infection in a preclinical model.

Vectored immunoprophylaxis was first developed as a means of establishing engineered immunity to HIV using an adenoassociated viral vector expressing a broadly neutralizing antibody. We applied this concept to establish long-term prophylaxis against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a mouse model using adenoassociated virus and lentiviral vectors expressing a high-affinity angiotensin-converting enzyme 2 (ACE2) decoy. Administration of decoy-expressing (adenoassociated virus) AAV2.retro and AAV6.2 vectors by intranasal instillation or intramuscular injection protected mice against high-titered SARS-CoV-2 infection. AAV and lentiviral vectored immunoprophylaxis was durable and was active against SARS-CoV-2 Omicron subvariants. The AAV vectors were also effective therapeutically when administered postinfection. Vectored immunoprophylaxis could be of value for immunocompromised individuals for whom vaccination is not practical and as a means to rapidly establish protection from infection. Unlike monoclonal antibody therapy, the approach is expected to remain active despite continued evolution viral variants.

Rapidly Progressive Glomerulonephritis: A COVID-19 Case Report.

Anti-neutrophil cytoplasmic antibody (ANCA) associated vasculitis is a systemic autoimmune disease that typically presents as a multi-organ manifesting disease of unclear etiology that can predispose to rapidly progressive glomerulonephritis (RPGN). If left untreated, ANCA-associated vasculitis can be fatal, and RPGN can progress to irreversible renal failure. Environmental and genetic factors have been implicated in the pathogenesis of this vasculitis. Coronavirus disease (COVID-19) has been noted to have various physiologic impacts on the body, with literature indicating possible autoimmune effects. We present a rare case of ANCA-associated vasculitis in an elderly male with no known autoimmune history after a recent illness with COVID-19. The patient had been seen as an outpatient with progressively declining renal function until he presented to the hospital with acute renal failure and pericarditis. Workup revealed elevated anti-myeloperoxidase antibody (MPO-AB) and perinuclear ANCA (p-ANCA) antibodies with a biopsy confirming focal cresenteric glomerulonephritis, and the patient was initiated on steroid therapy with notable improvement and a return to baseline kidney function.

Single-shot AAV-vectored vaccine against SARS-CoV-2 with fast and long-lasting immunity.

Due to the insufficient long-term protection and significant efficacy reduction to new variants of current COVID-19 vaccines, the epidemic prevention and control are still challenging. Here, we employ a capsid and antigen structure engineering (CASE) strategy to manufacture an adeno-associated viral serotype 6-based vaccine (S663V-RBD), which expresses trimeric receptor binding domain (RBD) of spike protein fused with a biological adjuvant RS09. Impressively, the engineered S663V-RBD could rapidly induce a satisfactory RBD-specific IgG titer within 2 weeks and maintain the titer for more than 4 months. Compared to the licensed BBIBP-CorV (Sinopharm, China), a single-dose S663V-RBD induced more endurable and robust immune responses in mice and elicited superior neutralizing antibodies against three typical SARS-CoV-2 pseudoviruses including wild type, C.37 (Lambda) and B.1.617.2 (Delta). More interestingly, the intramuscular injection of S663V-RBD could overcome pre-existing immunity against the capsid. Given its effectiveness, the CASE-based S663V-RBD may provide a new solution for the current and next pandemic.

The HAVEN study-hydroxychloroquine in ANCA vasculitis evaluation-a multicentre, randomised, double-blind, placebo-controlled trial: study protocol and statistical analysis plan.

BACKGROUND: Patients with non-severe ANCA-associated vasculitis (AAV) are often prescribed immunosuppressive medications that are associated with severe side effects and a reduced quality of life. There is an unmet need for safer effective treatments for these patients. Hydroxychloroquine is being explored due to its effect in similar autoimmune conditions such as systemic lupus erythematosus. METHODS: Double-blind, placebo-controlled multicentre trial recruiting 76 patients across 20 sites. Participants will be randomised 1:1 to hydroxychloroquine or placebo in addition to standard of care immunosuppressive therapies over the course of 52 weeks. A phase II selection design will be used to determine hdroxychloroquine's efficacy, using prednisolone dosage and Birmingham Vasculitis Activity Score as a measure of disease activity. Secondary outcomes will explore other elements of AAV progression, including disease flares and time to remission. DISCUSSION: This trial aims to explore Hydroxychloroquine as a treatment for patients with AAV. If effective, the need for immunosuppressive treatments such as prednisolone could be reduced. Hydroxychloroquine is safer, cheaper and has fewer adverse effects than conventional immunosuppressive treatments. This could improve patient outcomes while saving money for the NHS. TRIAL REGISTRATION: ISRCTN: ISRCTN79334891. Registered 07 June 2021. EudraCT: 2018-001268-40. Registered 13 September 2019. CLINICALTRIALS: gov: NCT04316494. Registered 20 March 2020.

Management of Antineutrophil Cytoplasmic Antibody-Associated Vasculitis with COVID-19: A Single Center Experience.

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) patients particularly presenting as rapidly progressive glomerulonephritis (RPGN) are at extremely high risk of progressing to end-stage kidney disease (ESKD); therefore, timely intervention is important. We describe our experience of managing six AAV patients who were on treatment (induction phase) and developed COVID-19. Cyclophosphamide was stopped till RT-PCR for SARS-CoV-2 was reported negative and patient had improved symptomatically. Out of our six patients, one died. Subsequently, cyclophosphamide was successfully resumed in all the surviving patients. In patients of AAV with COVID-19, close monitoring and withholding of cytotoxic medication and continuing steroids till active infection subsides is an effective treatment strategy until more and more data from well-conducted largescale studies become available for guidance.

Boosters for adeno-associated virus vector (AAV) (r)evolution.

Adeno-associated virus (AAV) is one of the most exciting and most versatile templates for engineering of gene-delivery vectors for use in human gene therapy, owing to the existence of numerous naturally occurring capsid variants and their amenability to directed molecular evolution. As a result, the field has witnessed an explosion of novel "designer" AAV capsids and ensuing vectors over the last two decades, which have been isolated from comprehensive capsid libraries generated through technologies such as DNA shuffling or peptide display, and stratified under stringent positive and/or negative selection pressures. Here, we briefly highlight a panel of recent, innovative and transformative methodologies that we consider to have exceptional potential to advance directed AAV capsid evolution and to thereby accelerate AAV vector revolution. These avenues comprise original technologies for (i) barcoding and high-throughput screening of individual AAV variants or entire capsid libraries, (ii) selection of transduction-competent AAV vectors on the DNA level, (iii) enrichment of expression-competent AAV variants on the RNA level, as well as (iv) high-resolution stratification of focused AAV capsid libraries on the single-cell level. Together with other emerging AAV engineering stratagems, such as rational design or machine learning, these pioneering techniques promise to provide an urgently needed booster for AAV (r)evolution.

Progress in Respiratory Gene Therapy.

The prospect of gene therapy for inherited and acquired respiratory disease has energized the research community since the 1980s, with cystic fibrosis, as a monogenic disorder, driving early efforts to develop effective strategies. The fact that there are still no approved gene therapy products for the lung, despite many early phase clinical trials, illustrates the scale of the challenge: In the 1990s, first-generation non-viral and viral vector systems demonstrated proof-of-concept but low efficacy. Since then, there has been steady progress toward improved vectors with the capacity to overcome at least some of the formidable barriers presented by the lung. In addition, the inclusion of features such as codon optimization and promoters providing long-term expression have improved the expression characteristics of therapeutic transgenes. Early approaches were based on gene addition, where a new DNA copy of a gene is introduced to complement a genetic mutation: however, the advent of RNA-based products that can directly express a therapeutic protein or manipulate gene expression, together with the expanding range of tools for gene editing, has stimulated the development of alternative approaches. This review discusses the range of vector systems being evaluated for lung delivery; the variety of cargoes they deliver, including DNA, antisense oligonucleotides, messenger RNA (mRNA), small interfering RNA (siRNA), and peptide nucleic acids; and exemplifies progress in selected respiratory disease indications.

A link between severe hepatitis in children and adenovirus 41 and adeno-associated virus 2 infections.

Over the past few months there have been reports of severe acute hepatitis in several hundred, otherwise healthy, immunocompetent young children. Several deaths have been recorded and a relatively large proportion of the patients have needed liver transplants. Most of the cases, so far, have been seen in the UK and in North America, but it has also been reported in many other European countries, the Middle East and Asia. Most common viruses have been ruled out as a causative agent; hepatitis A virus (HAV), hepatitis B virus (HBV) and hepatitis C virus (HCV) were not detected, nor were Epstein-Barr virus (EBV), cytomegalovirus (CMV) and human immunodeficiency virus (HIV) in many cases. A small proportion of the children had been infected with SARS-CoV-2 but these seem to be in a minority; similarly, almost none of the children had been vaccinated against COVID-19. Significantly, many of the patients were infected with adenovirus 41 (HAdV-F41). Previously, HAdV-41 had not been linked to hepatitis and is usually considered to cause gastroenteritis in both immunocompetent and immunocompromised patients. In two most recent studies, adeno-associated virus 2 (AAV2) was detected in almost all patients, together with species C and F HAdVs and human herpesvirus 6B (HHV6B). Here, I discuss the possibility that a change in tropism of HAdV-41 and changes in AAV2 may be responsible for their links to acute hepatitis.

Antineutrophil Cytoplasmic Antibody (ANCA)-Associated Renal Vasculitis Following COVID-19 Vaccination: A Case Report and Literature Review.

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is an immune-mediated disorder of small and medium-sized vessels, characterized by the production of autoantibodies that target the neutrophilic antigens leading to mononuclear cell infiltration and destruction of blood vessels in lungs, skin, and kidneys. Although rare, the coronavirus disease 2019 (COVID-19) vaccine may trigger autoimmune vasculitis. We report a rare case of ANCA-associated renal vasculitis following COVID-19 vaccination in a 59-year-old male who presented with flu-like symptoms and deranged renal function tests. He received his second dose of the Pfizer COVID-19 vaccine 17 days ago. His clinical picture, serological testing, and radiological imaging were concerned with glomerular disease. His serum was positive for ANCAs, and the renal biopsy specimen revealed pauci-immune glomerulonephritis. He was diagnosed with AAV-associated renal vasculitis following COVID-19 vaccination because no other etiology was identified. His clinical improvement after starting rituximab and steroids reinforced the diagnosis.

Environmental factors influencing the risk of ANCA-associated vasculitis.

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a group of diseases characterized by inflammation and destruction of small and medium-sized blood vessels. Clinical disease phenotypes include microscopic polyangiitis (MPA), granulomatosis with polyangiitis (GPA), and eosinophilic granulomatosis with polyangiitis (EGPA). The incidence of AAV has been on the rise in recent years with advances in ANCA testing. The etiology and pathogenesis of AAV are multifactorial and influenced by both genetic and environmental factors, as well as innate and adaptive immune system responses. Multiple case reports have shown that sustained exposure to silica in an occupational environment resulted in a significantly increased risk of ANCA positivity. A meta-analysis involving six case-control studies showed that silica exposure was positively associated with AAV incidence. Additionally, exposure to air pollutants, such as carbon monoxide (CO), is a risk factor for AAV. AAV has seasonal trends. Studies have shown that various environmental factors stimulate the body to activate neutrophils and expose their own antigens, resulting in the release of proteases and neutrophil extracellular traps, which damage vascular endothelial cells. Additionally, the activation of complement replacement pathways may exacerbate vascular inflammation. However, the role of environmental factors in the etiology of AAV remains unclear and has received little attention. In this review, we summarized the recent literature on the study of environmental factors, such as seasons, air pollution, latitude, silica, and microbial infection, in AAV with the aim of exploring the relationship between environmental factors and AAV and possible mechanisms of action to provide a scientific basis for the prevention and treatment of AAV.

Rational design of AAVrh10-vectored ACE2 functional domain to broadly block the cell entry of SARS-CoV-2 variants.

The frequently emerging SARS-CoV-2 variants have weakened the effectiveness of existing COVID-19 vaccines and neutralizing antibody therapy. Nevertheless, the infections of SARS-CoV-2 variants still depend on angiotensin-converting enzyme 2 (ACE2) receptor-mediated cell entry, and thus the soluble human ACE2 (shACE2) is a potential decoy for broadly blocking SARS-CoV-2 variants. In this study, we firstly generated the recombinant AAVrh10-vectored shACE2 constructs, a kind of adeno-associated virus (AAV) serotype with pulmonary tissue tropism, and then validated its inhibition capacity against SARS-CoV-2 infection. To further optimize the minimized ACE2 functional domain candidates, a comprehensive analysis was performed to clarify the interactions between the ACE2 orthologs from various species and the receptor binding domain (RBD) of SARS-CoV-2 spike (S) protein. Based on the key interface amino acids, we designed a series of truncated ACE2 orthologs, and then assessed their potential affinity to bind to SARS-CoV-2 variants RBD in silico. Of note, we found that the 24-83aa fragment of dog ACE2 (dACE224-83) had a higher affinity to the RBD of SARS-CoV-2 variants than that of human ACE2. Importantly, AAVrh10-vectored shACE2 or dACE224-83 constructs exhibited a broadly blockage breadth against SARS-CoV-2 prototype and variants in vitro and ex vivo. Collectively, these data highlighted a promising therapeutic strategy against SARS-CoV-2 variants.

ANCA-associated vasculitis following Johnson and Johnson COVID-19 vaccine.

Introduction: Antineutrophil cytoplasmic autoantibodies associated vasculitis (AAV) is characterized by antibodies against antigens in cytoplasmic granules of neutrophils and predominantly affects small vessels. AAV after COVID-19 mRNA vaccination has been reported. Case presentation: We report a rare case of AAV in a patient who presented with rapidly progressive glomerulonephritis (RPGN) after Johnson & Johnson COVID-19 vaccine administration. Discussion: The temporal causal association between autoimmune manifestations like AAV and COVID-19 vaccines can be explained by hypothesized mechanisms like molecular mimicry, defective neutrophilic apoptosis, polyclonal activation, and systemic proinflammatory cytokine response. These mechanisms are likely to trigger autoimmune responses in genetically susceptible individuals. Still there are many research going on to fill the research gap on the development of ANCA associated with COVID-19 vaccines. Conclusion: Increasing reports of rare adverse effects like AAV following COVID-19 vaccination warrants the further study and evaluation of immune responses induced by those vaccines. Considering the potential severity of COVID-19 and the rarity of the above-mentioned adverse effects, COVID-19 vaccination should not be withheld.

Durable immunogenicity, adaptation to emerging variants, and low-dose efficacy of an AAV-based COVID-19 vaccine platform in macaques.

The COVID-19 pandemic continues to have devastating consequences on health and economy, even after the approval of safe and effective vaccines. Waning immunity, the emergence of variants of concern, breakthrough infections, and lack of global vaccine access and acceptance perpetuate the epidemic. Here, we demonstrate that a single injection of an adenoassociated virus (AAV)-based COVID-19 vaccine elicits at least 17-month-long neutralizing antibody responses in non-human primates at levels that were previously shown to protect from viral challenge. To improve the scalability of this durable vaccine candidate, we further optimized the vector design for greater potency at a reduced dose in mice and non-human primates. Finally, we show that the platform can be rapidly adapted to other variants of concern to robustly maintain immunogenicity and protect from challenge. In summary, we demonstrate this class of AAV can provide durable immunogenicity, provide protection at dose that is low and scalable, and be adapted readily to novel emerging vaccine antigens thus may provide a potent tool in the ongoing fight against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2).

Immunogenicity of an AAV-Based COVID-19 Vaccine in Murine Models of Obesity and Aging.

The SARS-CoV-2 pandemic has had a disastrous impact on global health. Although some vaccine candidates have been effective in combating SARS-CoV-2, logistical, economical, and sociological aspects still limit vaccine access globally. Recently, we reported on two room-temperature stable AAV-based COVID-19 vaccines that induced potent and protective immunogenicity following a single injection in murine and primate models. Obesity and old age are associated with increased mortality in COVID-19, as well as reduced immunogenicity and efficacy of vaccines. Here, we investigated the effectiveness of the AAVCOVID vaccine candidates in murine models of obesity and aging. Results demonstrate that obesity did not significantly alter the immunogenicity of either vaccine candidate. In aged mice, vaccine immunogenicity was impaired. These results suggest that AAV-based vaccines may have limitations in older populations and may be equally applicable in obese and non-obese populations.

An AAV vaccine targeting the RBD of the SARS-CoV-2 S protein induces effective neutralizing antibody titers in mice and canines.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has resulted in catastrophic damage worldwide. Accordingly, the development of powerful, safe, easily accessible vaccines with long-term effectiveness is understood as an urgently needed countermeasure against this ongoing pandemic. Guided by this strong promise of using AAVs, we here designed, optimized, and developed an AAV-based vaccines (including AAV-RBD(max), AAV-RBD(wt), AAV-2xRBD, and AAV-3xRBD) that elicit strong immune responses against the RBD domain of the SARS-CoV-2 S protein. These immunogenic responses have proven long-lived, with near peak levels for at least six months in mice. Notably, the sera immunized with AAV-3xRBD vaccine contains powerful neutralizing antibodies against the SARS-CoV-2 pseudovirus. Further evidence proven that potent specific antibodies could also be elicited in canines after vaccination with AAV-3xRBD vaccine.

Development of an Adeno-Associated Virus-Vectored SARS-CoV-2 Vaccine and Its Immunogenicity in Mice.

Owing to the outbreak of the novel coronavirus (SARS-CoV-2) worldwide at the end of 2019, the development of a SARS-CoV-2 vaccine became an urgent need. In this study, we developed a type 9 adeno-associated virus vectored vaccine candidate expressing a dimeric receptor binding domain (RBD) of the SARS-CoV-2 spike protein (S protein) and evaluated its immunogenicity in a murine model. The vaccine candidate, named AAV9-RBD virus, was constructed by inserting a signal peptide to the N-terminus of two copies of RBD, spaced by a linker, into the genome of a type 9 adeno-associated virus. In vitro assays showed that HeLa cells infected by the recombinant AAV virus expressed high levels of the recombinant RBD protein, mostly found in the cell culture supernatant. The recombinant AAV9-RBD virus was cultured and purified. The genome titer of the purified recombinant AAV9-RBD virus was determined to be 2.4 × 1013 genome copies/mL (GC/mL) by Q-PCR. Balb/c mice were immunized with the virus by intramuscular injection or nasal drip administration. Eight weeks after immunization, neutralizing antibodies against the new coronavirus pseudovirus were detected in the sera of all mice; the mean neutralizing antibody EC50 values were 517.7 ± 292.1 (n=10) and 682.8 ± 454.0 (n=10) in the intramuscular injection group and nasal drip group, respectively. The results of this study showed that the recombinant AAV9-RBD virus may be used for the development of a SARS-CoV-2 vaccine.

Novel Insights into the Therapeutic Potential of Lung-Targeted Gene Transfer in the Most Common Respiratory Diseases.

Over the past decades, a better understanding of the genetic and molecular alterations underlying several respiratory diseases has encouraged the development of new therapeutic strategies. Gene therapy offers new therapeutic alternatives for inherited and acquired diseases by delivering exogenous genetic materials into cells or tissues to restore physiological protein expression and/or activity. In this review, we review (1) different types of viral and non-viral vectors as well as gene-editing techniques; and (2) the application of gene therapy for the treatment of respiratory diseases and disorders, including pulmonary arterial hypertension, idiopathic pulmonary fibrosis, cystic fibrosis, asthma, alpha-1 antitrypsin deficiency, chronic obstructive pulmonary disease, non-small-cell lung cancer, and COVID-19. Further, we also provide specific examples of lung-targeted therapies and discuss the major limitations of gene therapy.

Induction of Hepatitis E Virus Anti-ORF3 Antibodies from Systemic Administration of a Muscle-Specific Adeno-Associated Virus (AAV) Vector.

The hepatitis E virus (HEV) is a major global health problem, leading to large outbreaks in the developing world and chronic infections in the developed world. HEV is a non-enveloped virus, which circulates in the blood in a quasi-enveloped form. The quasi-envelope protects HEV particles from neutralising anti-capsid antibodies in the serum; however, most vaccine approaches are designed to induce an immune response against the HEV capsid. In this study, we explored systemic in vivo administration of a novel synthetic and myotropic Adeno-associated virus vector (AAVMYO3) to express the small HEV phosphoprotein ORF3 (found on quasi-enveloped HEV) in the musculature of mice, resulting in the robust and dose-dependent formation of anti-ORF3 antibodies. Neutralisation assays using the serum of ORF3 AAV-transduced mice showed a modest inhibitory effect on the infection of quasi-enveloped HEV in vivo, comparable to previously characterised anti-ORF3 antibodies used as a control. The novel AAVMYO3 capsid used in this study can serve as a versatile platform for the continued development of vector-based vaccines against HEV and other infectious agents, which could complement traditional vaccines akin to the current positive experience with SARS-CoV-2.

Ex vivo and in vivo suppression of SARS-CoV-2 with combinatorial AAV/RNAi expression vectors.

Despite rapid development and deployment of vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), clinically relevant modalities to curb the pandemic by directly attacking the virus on a genetic level remain highly desirable and are urgently needed. Here we comprehensively illustrate the capacity of adeno-associated virus (AAV) vectors co-expressing a cocktail of three short hairpin RNAs (shRNAs; RNAi triggers) directed against the SARS-CoV-2 RdRp and N genes as versatile and effective antiviral agents. In cultured monkey cells and human gut organoids, our most potent vector, SAVIOR (SARS virus repressor), suppressed SARS-CoV-2 infection to background levels. Strikingly, in control experiments using single shRNAs, multiple SARS-CoV-2 escape mutants quickly emerged from infected cells within 24-48 h. Importantly, such adverse viral adaptation was fully prevented with the triple-shRNA AAV vector even during long-term cultivation. In addition, AAV-SAVIOR efficiently purged SARS-CoV-2 in a new model of chronically infected human intestinal cells. Finally, intranasal AAV-SAVIOR delivery using an AAV9 capsid moderately diminished viral loads and/or alleviated disease symptoms in hACE2-transgenic or wild-type mice infected with human or mouse SARS-CoV-2 strains, respectively. Our combinatorial and customizable AAV/RNAi vector complements ongoing global efforts to control the coronavirus disease 2019 (COVID-19) pandemic and holds great potential for clinical translation as an original and flexible preventive or therapeutic antiviral measure.

Non-invasive administration of AAV to target lung parenchymal cells and develop SARS-CoV-2-susceptible mice.

Adeno-associated virus (AAV)-mediated gene delivery holds great promise for gene therapy. However, the non-invasive delivery of AAV for lung tissues has not been adequately established. Here, we revealed that the intratracheal administration of an appropriate amount of AAV2/8 predominantly targets lung tissue. AAV-mediated gene delivery that we used in this study induced the expression of the desired protein in lung parenchymal cells, including alveolar type II cells. We harnessed the technique to develop severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-susceptible mice. Three kinds of immune function-relevant gene knockout (KO) mice were transduced with AAV encoding human angiotensin-converting enzyme 2 (hACE2) and then injected with SARS-CoV-2. Among these mice, type I interferon receptor (IFNAR) KO mice showed increased viral titer in the lungs compared to that in the other KO mice. Moreover, nucleocapsid protein of SARS-CoV-2 and multiple lesions in the trachea and lung were observed in AAV-hACE2-transduced, SARS-CoV-2-infected IFNAR KO mice, indicating the involvement of type I interferon signaling in the protection of SARS-CoV-2. In this study, we demonstrate the ease and rapidness of the intratracheal administration of AAV for targeting lung tissue in mice, and this can be used to study diverse pulmonary diseases.