Strategies to circumvent humoral immunity to adeno-associated viral vectors

Recent success in gene therapy of certain monogenic diseases in the clinic has infused enthusiasm into the continued development of recombinant adeno-associated viral (AAV) vectors as next-generation biologics. However, progress in clinical trials has also highlighted the challenges posed by the host humoral immune response to AAV vectors. Specifically, while pre-existing neutralizing antibodies (NAbs) limit the cohort of eligible patients, NAb generation following treatment prevents vector re-dosing

The Current and Future State of Vaccines, Antivirals and Gene Therapies Against Emerging Coronaviruses

Emerging coronaviruses (CoV) are constant global public health threats to society. Multiple ongoing clinical trials for vaccines and antivirals against CoVs showcase the availability of medical interventions to both prevent and treat the future emergence of highly pathogenic CoVs in human. However, given the diverse nature of CoVs and our close interactions with wild, domestic and companion animals, the next epidemic zoonotic CoV could resist the existing vaccines and antivirals developed, which are primarily focused on Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East Respiratory Syndrome Coronavirus (MERS CoV). In late 2019, the novel CoV (SARS-CoV-2) emerged in Wuhan, China, causing global public health concern. In this review, we will summarize the key advancements of current vaccines and antivirals against SARS-CoV and MERS-CoV as well as discuss the challenge and opportunity in the current SARS-CoV-2 crisis. At the end, we advocate the development of a “plug-and-play” platform technologies that could allow quick manufacturing and administration of broad-spectrum countermeasures in an outbreak setting. We will discuss the potential of AAV-based gene therapy technology for in vivo therapeutic antibody delivery to combat SARS-CoV-2 outbreak and the future emergence of severe CoVs

β-D-N4-hydroxycytidine (NHC) Inhibits SARS-CoV-2 Through Lethal Mutagenesis But Is Also Mutagenic To Mammalian Cells

Mutagenic ribonucleosides can act as broad-based antiviral agents. They are metabolized to the active ribonucleoside triphosphate form and concentrate in the genomes of RNA viruses during viral replication. β-D-N 4-hydroxycytidine (NHC, the initial metabolite of molnupiravir) is more than 100-fold more active than ribavirin or favipiravir against SARS-CoV-2, with antiviral activity correlated to the level of mutagenesis in virion RNA. However, NHC also displays host mutational activity in an animal cell culture assay, consistent with RNA and DNA precursors sharing a common intermediate of a ribonucleoside diphosphate. These results indicate that highly active mutagenic ribonucleosides may hold risk for the host

Target Site Recognition by a Diversity-Generating Retroelement

Diversity-generating retroelements (DGRs) are in vivo sequence diversification machines that are widely distributed in bacterial, phage, and plasmid genomes. They function to introduce vast amounts of targeted diversity into protein-encoding DNA sequences via mutagenic homing. Adenine residues are converted to random nucleotides in a retrotransposition process from a donor template repeat (TR) to a recipient variable repeat (VR). Using the Bordetella bacteriophage BPP-1 element as a prototype, we have characterized requirements for DGR target site function. Although sequences upstream of VR are dispensable, a 24 bp sequence immediately downstream of VR, which contains short inverted repeats, is required for efficient retrohoming. The inverted repeats form a hairpin or cruciform structure and mutational analysis demonstrated that, while the structure of the stem is important, its sequence can vary. In contrast, the loop has a sequence-dependent function. Structure-specific nuclease digestion confirmed the existence of a DNA hairpin/cruciform, and marker coconversion assays demonstrated that it influences the efficiency, but not the site of cDNA integration. Comparisons with other phage DGRs suggested that similar structures are a conserved feature of target sequences. Using a kanamycin resistance determinant as a reporter, we found that transplantation of the IMH and hairpin/cruciform-forming region was sufficient to target the DGR diversification machinery to a heterologous gene. In addition to furthering our understanding of DGR retrohoming, our results suggest that DGRs may provide unique tools for directed protein evolution via in vivo DNA diversification

Genomewide CRISPR knockout screen identified PLAC8 as an essential factor for SADS-CoVs infection

Zoonotic transmission of coronaviruses poses an ongoing threat to human populations. Endemic outbreaks of swine acute diarrhea syndrome coronavirus (SADS-CoV) have caused severe economic losses in the pig industry and have the potential to cause human outbreaks. Currently, there are no vaccines or specific antivirals against SADS-CoV, and our limited understanding of SADS-CoV host entry factors could hinder prompt responses to a potential human outbreak. Using a genomewide CRISPR knockout screen, we identified placenta-associated 8 protein (PLAC8) as an essential host factor for SADS-CoV infection. Knockout of PLAC8 abolished SADS-CoV infection, which was restored by complementing PLAC8 from multiple species, including human, rhesus macaques, mouse, pig, pangolin, and bat, suggesting a conserved infection pathway and susceptibility of SADS-CoV among mammals. Mechanistically, PLAC8 knockout does not affect viral entry; rather, knockout cells displayed a delay and reduction in viral subgenomic RNA expression. In a swine primary intestinal epithelial culture (IEC) infection model, differentiated cultures have high levels of PLAC8 expression and support SADS-CoV replication. In contrast, expanding IECs have low levels of PLAC8 expression and are resistant to SADS-CoV infection. PLAC8 expression patterns translate in vivo; the immunohistochemistry of swine ileal tissue revealed high levels of PLAC8 protein in neonatal compared to adult tissue, mirroring the known SADS-CoV pathogenesis in neonatal piglets. Overall, PLAC8 is an essential factor for SADS-CoV infection and may serve as a promising target for antiviral development for potential pandemic SADS-CoV

The receptor binding domain of the viral spike protein is an immunodominant and highly specific target of antibodies in SARS-CoV-2 patients

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that first emerged in late 2019 is responsible for a pandemic of severe respiratory illness. People infected with this highly contagious virus can present with clinically inapparent, mild, or severe disease. Currently, the virus infection in individuals and at the population level is being monitored by PCR testing of symptomatic patients for the presence of viral RNA. There is an urgent need for SARS-CoV-2 serologic tests to identify all infected individuals, irrespective of clinical symptoms, to conduct surveillance and implement strategies to contain spread. As the receptor binding domain (RBD) of the spike protein is poorly conserved between SARS-CoVs and other pathogenic human coronaviruses, the RBD represents a promising antigen for detecting CoV-specific antibodies in people. Here we use a large panel of human sera (63 SARS-CoV-2 patients and 71 control subjects) and hyperimmune sera from animals exposed to zoonotic CoVs to evaluate RBD's performance as an antigen for reliable detection of SARS-CoV-2-specific antibodies. By day 9 after the onset of symptoms, the recombinant SARS-CoV-2 RBD antigen was highly sensitive (98%) and specific (100%) for antibodies induced by SARS-CoVs. We observed a strong correlation between levels of RBD binding antibodies and SARS-CoV-2 neutralizing antibodies in patients. Our results, which reveal the early kinetics of SARS-CoV-2 antibody responses, support using the RBD antigen in serological diagnostic assays and RBD-specific antibody levels as a correlate of SARS-CoV-2 neutralizing antibodies in people

IgG-like bispecific antibodies with potent and synergistic neutralization against circulating SARS-CoV-2 variants of concern

Monoclonal antibodies are a promising approach to treat COVID-19, however the emergence of SARS-CoV-2 variants has challenged the efficacy and future of these therapies. Antibody cocktails are being employed to mitigate these challenges, but neutralization escape remains a major challenge and alternative strategies are needed. Here we present two anti-SARS-CoV-2 spike binding antibodies, one Class 1 and one Class 4, selected from our non-immune human single-chain variable fragment (scFv) phage library, that are engineered into four, fully-human IgG-like bispecific antibodies (BsAb). Prophylaxis of hACE2 mice and post-infection treatment of golden hamsters demonstrates the efficacy of the monospecific antibodies against the original Wuhan strain, while promising in vitro results with the BsAbs demonstrate enhanced binding and distinct synergistic effects on neutralizing activity against circulating variants of concern. In particular, one BsAb engineered in a tandem scFv-Fc configuration shows synergistic neutralization activity against several variants of concern including B.1.617.2. This work provides evidence that synergistic neutralization can be achieved using a BsAb scaffold, and serves as a foundation for the future development of broadly reactive BsAbs against emerging variants of concern

SARS-CoV-2 D614G variant exhibits efficient replication ex vivo and transmission in vivo

The spike D614G substitution is prevalent in global SARS-CoV-2 strains, but its effects on viral pathogenesis and transmissibility remain unclear. We engineered a SARS-CoV-2 variant containing this substitution. The variant exhibits more efficient infection, replication, and competitive fitness in primary human airway epithelial cells, but maintains similar morphology and in vitro neutralization properties, compared with the ancestral wild-type virus. Infection of human angiotensin-converting enzyme 2 (ACE2) transgenic mice and Syrian hamsters with both viruses resulted in similar viral titers in respiratory tissues and pulmonary disease. However, the D614G variant transmits significantly faster and displayed increased competitive fitness than the wild-type virus in hamsters. These data show that the D614G substitution enhances SARS-CoV-2 infectivity, competitive fitness, and transmission in primary human cells and animal models

Repurposing the Ebola and Marburg Virus Inhibitors Tilorone, Quinacrine, and Pyronaridine: In Vitro Activity against SARS-CoV-2 and Potential Mechanisms

Severe acute respiratory coronavirus 2 (SARS-CoV-2) is a newly identified virus that has resulted in over 2.5 million deaths globally and over 116 million cases globally in March, 2021. Small-molecule inhibitors that reverse disease severity have proven difficult to discover. One of the key approaches that has been widely applied in an effort to speed up the translation of drugs is drug repurposing. A few drugs have shown in vitro activity against Ebola viruses and demonstrated activity against SARS-CoV-2 in vivo. Most notably, the RNA polymerase targeting remdesivir demonstrated activity in vitro and efficacy in the early stage of the disease in humans. Testing other small-molecule drugs that are active against Ebola viruses (EBOVs) would appear a reasonable strategy to evaluate their potential for SARS-CoV-2. We have previously repurposed pyronaridine, tilorone, and quinacrine (from malaria, influenza, and antiprotozoal uses, respectively) as inhibitors of Ebola and Marburg viruses in vitro in HeLa cells and mouse-adapted EBOV in mice in vivo. We have now tested these three drugs in various cell lines (VeroE6, Vero76, Caco-2, Calu-3, A549-ACE2, HUH-7, and monocytes) infected with SARS-CoV-2 as well as other viruses (including MHV and HCoV 229E). The compilation of these results indicated considerable variability in antiviral activity observed across cell lines. We found that tilorone and pyronaridine inhibited the virus replication in A549-ACE2 cells with IC50 values of 180 nM and IC50 198 nM, respectively. We used microscale thermophoresis to test the binding of these molecules to the spike protein, and tilorone and pyronaridine bind to the spike receptor binding domain protein with Kd values of 339 and 647 nM, respectively. Human Cmax for pyronaridine and quinacrine is greater than the IC50 observed in A549-ACE2 cells. We also provide novel insights into the mechanism of these compounds which is likely lysosomotropic

Targeted isolation of diverse human protective broadly neutralizing antibodies against SARS-like viruses

The emergence of current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) and potential future spillovers of SARS-like coronaviruses into humans pose a major threat to human health and the global economy. Development of broadly effective coronavirus vaccines that can mitigate these threats is needed. Here, we utilized a targeted donor selection strategy to isolate a large panel of human broadly neutralizing antibodies (bnAbs) to sarbecoviruses. Many of these bnAbs are remarkably effective in neutralizing a diversity of sarbecoviruses and against most SARS-CoV-2 VOCs, including the Omicron variant. Neutralization breadth is achieved by bnAb binding to epitopes on a relatively conserved face of the receptor-binding domain (RBD). Consistent with targeting of conserved sites, select RBD bnAbs exhibited protective efficacy against diverse SARS-like coronaviruses in a prophylaxis challenge model in vivo. These bnAbs provide new opportunities and choices for next-generation antibody prophylactic and therapeutic applications and provide a molecular basis for effective design of pan-sarbecovirus vaccines