Antigenic characterisation of human monoclonal antibodies for therapeutic use against H7N9 avian influenza virus (preprint)

ABSTRACT Since 2013, H7N9 avian influenza viruses (AIVs) have caused more than 1500 human deaths and millions of poultry culling. Despite large-scale poultry vaccination, H7N9 AIVs continue to circulate among poultry in China and pose a threat to human health. Previously, we isolated and generated four monoclonal antibodies (mAbs) derived from humans naturally infected with H7N9 AIV. Here, we investigated the haemagglutinin (HA) epitopes of H7N9 AIV targeted by these mAbs (L3A-44, K9B-122, L4A-14 and L4B-18) using immune escape studies. Our results revealed four key antigenic epitopes at HA amino acid positions 125, 133, 149, and 217. The mutant H7N9 viruses representing escape mutations containing Alanine to Threonine at residue 125 (A125T), Glycine to Glutamic acid at residue 133 (G133E), Asparagine to Aspartic acid at residue 149 (N149D), or Leucine to Glutamine at residue 217 (L217Q) showed reduced or completely abolished cross-reactivity with the mAbs, as measured by hemagglutination inhibition (HI) assay. We further assessed the potential risk of these mutants to humans should they emerge following mAb treatment by measuring the impact of these HA mutations on virus fitness and evasion of host adaptive immunity. Here we showed that the L4A-14 mAb had broad neutralizing capability, and its escape mutant N149D had reduced viral stability and human receptor binding and could be neutralized by both post-infection and antigen-induced sera. Therefore, L4A-14 mAb could be a therapeutic candidate for H7N9 AIV infection in humans and warrants further investigation for therapeutic application. IMPORTANCE Avian Influenza virus (AIV) H7N9 continues to circulate and evolve in birds, posing a credible threat to humans. Antiviral drugs have been proven useful for the treatment of severe influenza infections in humans, however, concerns have been raised as antiviral resistant mutants have emerged. Monoclonal antibodies (mAbs) have been studied for both prophylactic and therapeutic applications in infectious disease control and have demonstrated great potential. For example, mAb treatment has significantly reduced the risk of people developing severe disease with SARS-COV 2 infection. In addition to the protection efficiency, we should also consider the potential risk of the escape mutants generated by mAb treatment to public health by assessing their viral fitness and potential to compromise host adaptive immunity. Considering these parameters, we assessed four human mAbs derived from humans naturally infected with H7N9 AIV and showed that the mAb L4A-14 displayed potential as a therapeutic candidate.

A COVID-19 vaccine candidate using SpyCatcher multimerization of the SARS-CoV-2 spike protein receptor-binding domain induces potent neutralising antibody responses (preprint)

There is dire need for an effective and affordable vaccine against SARS-CoV-2 to tackle the ongoing pandemic. In this study, we describe a modular virus-like particle vaccine candidate displaying the SARS-CoV-2 spike glycoprotein receptor-binding domain (RBD) using SpyTag/SpyCatcher technology (RBD-SpyVLP). Low doses of RBD-SpyVLP in a prime-boost regimen induced a strong neutralising antibody response in mice and pigs that was superior to convalescent human sera. We evaluated antibody quality using ACE2 blocking and neutralisation of cell infection by pseudovirus or wild-type SARS-CoV-2. Using competition assays with a monoclonal antibody panel, we showed that RBD-SpyVLP induced a polyclonal antibody response that recognised all key epitopes on the RBD, reducing the likelihood of selecting neutralisation-escape mutants. The induction of potent and polyclonal antibody responses by RBD-SpyVLP provides strong potential to address clinical and logistic challenges of the COVID-19 pandemic. Moreover, RBD-SpyVLP is highly resilient, thermostable and can be lyophilised without losing immunogenicity, to facilitate global distribution and reduce cold-chain dependence.

Structure-Altering Mutations of the SARS-CoV-2 Frame Shifting RNA Element (preprint)

With the rapid rate of Covid-19 infections and deaths, treatments and cures besides hand washing, social distancing, masks, isolation, and quarantines are urgently needed. The treatments and vaccines rely on the basic biophysics of the complex viral apparatus. While proteins are serving as main drug and vaccine targets, therapeutic approaches targeting the 30,000 nucleotide RNA viral genome form important complementary approaches. Indeed, the high conservation of the viral genome, its close evolutionary relationship to other viruses, and the rise of gene editing and RNA-based vaccines all argue for a focus on the RNA agent itself. One of the key steps in the viral replication cycle inside host cells is the ribosomal frameshifting required for translation of overlapping open reading frames. The frameshifting element (FSE), one of three highly conserved regions of coronaviruses, includes an RNA pseudoknot considered essential for this ribosomal switching. In this work, we apply our graph-theory-based framework for representing RNA secondary structures, "RAG" (RNA-As Graphs), to alter key structural features of the FSE of the SARS-CoV-2 virus. Specifically, using RAG machinery of genetic algorithms for inverse folding adapted for RNA structures with pseudoknots, we computationally predict minimal mutations that destroy a structurally-important stem and/or the pseudoknot of the FSE, potentially dismantling the virus against translation of the polyproteins. Additionally, our microsecond molecular dynamics simulations of mutant structures indicate relatively stable secondary structures. These findings not only advance our computational design of RNAs containing pseudoknots; they pinpoint to key residues of the SARS-CoV-2 virus as targets for anti-viral drugs and gene editing approaches. SIGNIFICANCESince the outbreak of Covid-19, numerous projects were launched to discover drugs and vaccines. Compared to protein-focused approaches, targeting the RNA genome, especially highly conserved crucial regions, can destruct the virus life cycle more fundamentally and avoid problems of viral mutations. We choose to target the small frame-shifting element (FSE) embedded in the Open Reading Frame 1a,b of SARS-CoV-2. This FSE is essential for translating overlapping reading frames and thus controlling the viral protein synthesis pathway. By applying graph-theory-based computational algorithms, we identify structurally crucial residues in the FSE as potential targets for anti-viral drugs and gene editing.

Evaluation of the immunogenicity of prime-boost vaccination with the replication-deficient viral vectored COVID-19 vaccine candidate ChAdOx1 nCoV-19 (preprint)

Clinical development of the COVID-19 vaccine candidate ChAdOx1 nCoV-19, a replication-deficient simian adenoviral vector expressing the full-length SARS-CoV-2 spike (S) protein was initiated in April 2020 following non-human primate studies using a single immunisation. Here, we compared the immunogenicity of one or two doses of ChAdOx1 nCoV-19 in both mice and pigs. Whilst a single dose induced antigen-specific antibody and T cells responses, a booster immunisation enhanced antibody responses, particularly in pigs, with a significant increase in SARS-CoV-2 neutralising titres.

The SARS-CoV-2 Spike protein has a broad tropism for mammalian ACE2 proteins (preprint)

SARS-CoV-2 emerged in late 2019, leading to the COVID-19 pandemic that continues to cause significant global mortality in human populations. Given its sequence similarity to SARS-CoV, as well as related coronaviruses circulating in bats, SARS-CoV-2 is thought to have originated in Chiroptera species in China. However, whether the virus spread directly to humans or through an intermediate host is currently unclear, as is the potential for this virus to infect companion animals, livestock and wildlife that could act as viral reservoirs. Using a combination of surrogate entry assays and live virus we demonstrate that, in addition to human ACE2, the Spike glycoprotein of SARS-CoV-2 has a broad host tropism for mammalian ACE2 receptors, despite divergence in the amino acids at the Spike receptor binding site on these proteins. Of the twenty-two different hosts we investigated, ACE2 proteins from dog, cat and rabbit were the most permissive to SARS-CoV-2, while bat and bird ACE2 proteins were the least efficiently used receptors. The absence of a significant tropism for any of the three genetically distinct bat ACE2 proteins we examined indicates that SARS-CoV-2 receptor usage likely shifted during zoonotic transmission from bats into people, possibly in an intermediate reservoir. Interestingly, while SARS-CoV-2 pseudoparticle entry was inefficient in cells bearing the ACE2 receptor from bats or birds the live virus was still able to enter these cells, albeit with markedly lower efficiency. The apparently broad tropism of SARS-CoV-2 at the point of viral entry confirms the potential risk of infection to a wide range of companion animals, livestock and wildlife.