Circumventing anti-vector immunity towards adenoviral vectored vaccines

Replication deficient (RD) adenoviruses (Ad) are the most widely administered viral vectors, with licensed SARS-CoV-2 vaccines using vectors derived from human Ad type 5 (Ad5) and 26 (Ad26), and chimpanzee Ad ''ChAdOx1''. Ad vectored vaccines generate robust cellular and humoral immunity, against both the transgene-encoded protein and the Ad vector itself. It's unclear how many times a single Ad vector can be readministered before this anti-vector immunity impairs generation of the desired transgene-specific adaptive responses. Antivector immunity also arises from naturally acquired Ad infections. In the absence of anti-Ad5 immunity, Ad5 is a goldstandard vector with robust vaccine immunogenicity, however widespread Ad5 seroprevalence hampers its use as a vector for the global population. We developed novel pseudotyped Ads as RD vectored vaccines encoding SARS-CoV-2 spike protein. These vectors exhibit fiber knob swaps from low seroprevalence Ads grafted onto an Ad5 backbone. We characterised innate immune responses following administration of these vectors in mice, and spikespecific adaptive responses three weeks later. Furthermore, we quantified the effects of anti-vector humoral immunity against these vectors in an in vitro transduction assay using human plasma. The pseudotyped vectors exhibit many desirable vaccine characteristics as the equivalent Ad5 vector, including CD4+ and CD8+ T cell responses against multiple spike epitopes. Importantly, fiber knob pseudotyping can substantially circumvent the direct, humoral, anti-vector immunity induced through Ad exposure in humans. These data indicate the fiber knob plays an important role in anti-vector immunity, and can be manipulated for evasion of such responses without hampering vaccine immunogenicity.

Loss of furin site enhances SARS-CoV-2 spike protein pseudovirus infection.

BACKGROUND: SARS-CoV-2 has a significant impact on healthcare systems all around the world. Due to its high pathogenicity, live SARS-CoV-2 must be handled under biosafety level 3 conditions. Pseudoviruses are useful virological tools because of their safety and versatility, but the low titer of these viruses remains a limitation for their more comprehensive applications. METHOD: Here, we constructed a Luc/eGFP based on a pseudotyped lentiviral HIV-1 system to transduce SARS-CoV-2 S glycoprotein to detect cell entry properties and cellular tropism. RESULTS: The furin cleavage site deletion of the S protein removed (SFko) can help SARS-CoV-2 S to be cleaved during viral packaging to improve infection efficiency. The furin cleavage site in SARS-CoV-2-S mediates membrane fusion and SFko leads to an increased level of S protein and limits S1/S2 cleavage to enhance pseudovirus infection in cells. Full-length S (SFL) pseudotyped with N, M, and E helper packaging can effectively help SFL infect cells. Finally, pseudotyped SFko particles were successfully used to detect neutralizing antibodies in RBD protein-immunized mouse serum. CONCLUSION: Overall, our study indicates a series of modifications that result in the production of relatively high-titer SARS-COV-2 pseudo-particles that may be suitable for the detection of neutralizing antibodies from COVID-19 patients.

Development of an efficient reproducible cell-cell transmission assay for rapid quantification of SARS-CoV-2 spike interaction with hACE2.

Efficient quantitative assays for measurement of viral replication and infectivity are indispensable for future endeavors to develop prophylactic or therapeutic antiviral drugs or vaccines against SARS-CoV-2. We developed a SARS-CoV-2 cell-cell transmission assay that provides a rapid and quantitative readout to assess SARS-CoV-2 spike hACE2 interaction in the absence of pseudotyped particles or live virus. We established two well-behaved stable cell lines, which demonstrated a remarkable correlation with standard cell-free viral pseudotyping for inhibition by convalescent sera, small-molecule drugs, and murine anti-spike monoclonal antibodies. The assay is rapid, reliable, and highly reproducible, without a requirement for any specialized research reagents or laboratory equipment and should be easy to adapt for use in most investigative and clinical settings. It can be effectively used or modified for high-throughput screening for compounds and biologics that interfere with virus-cell binding and entry to complement other neutralization assays currently in use.

Iota-Carrageenan Inhibits Replication of SARS-CoV-2 and the Respective Variants of Concern Alpha, Beta, Gamma and Delta.

The COVID-19 pandemic continues to spread around the world and remains a major public health threat. Vaccine inefficiency, vaccination breakthroughs and lack of supply, especially in developing countries, as well as the fact that a non-negligible part of the population either refuse vaccination or cannot be vaccinated due to age, pre-existing illness or non-response to existing vaccines intensify this issue. This might also contribute to the emergence of new variants, being more efficiently transmitted, more virulent and more capable of escaping naturally acquired and vaccine-induced immunity. Hence, the need of effective and viable prevention options to reduce viral transmission is of outmost importance. In this study, we investigated the antiviral effect of iota-, lambda- and kappa-carrageenan, sulfated polysaccharides extracted from red seaweed, on SARS-CoV-2 Wuhan type and the spreading variants of concern (VOCs) Alpha, Beta, Gamma and Delta. Carrageenans as part of broadly used nasal and mouth sprays as well as lozenges have the potential of first line defense to inhibit the infection and transmission of SARS-CoV-2. Here, we demonstrate by using a SARS-CoV-2 spike pseudotyped lentivirus particles (SSPL) system and patient-isolated SARS-CoV-2 VOCs to infect transgenic A549ACE2/TMPRSS2 and Calu-3 human lung cells that all three carrageenan types exert antiviral activity. Iota-carrageenan exhibits antiviral activity with comparable IC50 values against the SARS-CoV-2 Wuhan type and the VOCs. Altogether, these results indicate that iota-carrageenan might be effective for prophylaxis and treatment of SARS-CoV-2 infections independent of the present and potentially future variants.

SARS CoV-2 Nucleoprotein Enhances the Infectivity of Lentiviral Spike Particles.

The establishment of SARS CoV-2 spike-pseudotyped lentiviral (LV) systems has enabled the rapid identification of entry inhibitors and neutralizing agents, alongside allowing for the study of this emerging pathogen in BSL-2 level facilities. While such frameworks recapitulate the cellular entry process in ACE2+ cells, they are largely unable to factor in supplemental contributions by other SARS CoV-2 genes. To address this, we performed an unbiased ORF screen and identified the nucleoprotein (N) as a potent enhancer of spike-pseudotyped LV particle infectivity. We further demonstrate that the spike protein is better enriched in virions when the particles are produced in the presence of N protein. This enrichment of spike renders LV particles more infectious as well as less vulnerable to the neutralizing effects of a human IgG-Fc fused ACE2 microbody. Importantly, this improvement in infectivity is observed with both wild-type spike protein as well as the D614G mutant. Our results hold important implications for the design and interpretation of similar LV pseudotyping-based studies.

Effective Inhibition of SARS-CoV-2 Entry by Heparin and Enoxaparin Derivatives.

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has caused a pandemic of historic proportions and continues to spread globally, with enormous consequences to human health. Currently there is no vaccine, effective therapeutic, or prophylactic. As with other betacoronaviruses, attachment and entry of SARS-CoV-2 are mediated by the spike glycoprotein (SGP). In addition to its well-documented interaction with its receptor, human angiotensin-converting enzyme 2 (hACE2), SGP has been found to bind to glycosaminoglycans like heparan sulfate, which is found on the surface of virtually all mammalian cells. Here, we pseudotyped SARS-CoV-2 SGP on a third-generation lentiviral (pLV) vector and tested the impact of various sulfated polysaccharides on transduction efficiency in mammalian cells. The pLV vector pseudotyped SGP efficiently and produced high titers on HEK293T cells. Various sulfated polysaccharides potently neutralized pLV-S pseudotyped virus with clear structure-based differences in antiviral activity and affinity to SGP. Concentration-response curves showed that pLV-S particles were efficiently neutralized by a range of concentrations of unfractionated heparin (UFH), enoxaparin, 6-O-desulfated UFH, and 6-O-desulfated enoxaparin with 50% inhibitory concentrations (IC50s) of 5.99 µg/liter, 1.08 mg/liter, 1.77 µg/liter, and 5.86 mg/liter, respectively. In summary, several sulfated polysaccharides show potent anti-SARS-CoV-2 activity and can be developed for prophylactic as well as therapeutic purposes.IMPORTANCE The emergence of severe acute respiratory syndrome coronavirus (SARS-CoV-2) in Wuhan, China, in late 2019 and its subsequent spread to the rest of the world has created a pandemic situation unprecedented in modern history. While ACE2 has been identified as the viral receptor, cellular polysaccharides have also been implicated in virus entry. The SARS-CoV-2 spike glycoprotein (SGP) binds to glycosaminoglycans like heparan sulfate, which is found on the surface of virtually all mammalian cells. Here, we report structure-based differences in antiviral activity and affinity to SGP for several sulfated polysaccharides, including both well-characterized FDA-approved drugs and novel marine sulfated polysaccharides, which can be developed for prophylactic as well as therapeutic purposes.

Comparison of potency assays to assess SARS-CoV-2 neutralizing antibody capacity in COVID-19 convalescent plasma.

Convalescent plasma is plasma collected from individuals after resolution of an infection and the development of antibodies. Passive antibody administration by transfusion of convalescent plasma is currently in clinical evaluations to treat COVID-19 patients. The level of neutralizing antibodies vary among convalescent patients and fast and simple methods to identify suitable plasma donations are needed. We compared three methods to determine the SARS-CoV-2 neutralizing activity of human convalescent plasma: life virus neutralization by plaque reduction assay, a lentiviral vector based pseudotype neutralization assay and a competition ELISA-based surrogate virus neutralization assay (sVNT). Neutralization activity correlated among the different assays; however the sVNT assay was overvaluing the low neutralizing plasma. On the other hand, the sVNT assay required the lowest biosafety level, is fast and is sufficient to identify highly neutralizing plasma samples. Though weakly neutralizing samples were more reliable detected by the more challenging lentiviral vector based assays or virus neutralization assays. Spike receptor binding competition assays are suitable to identify highly neutralizing plasma samples under low biosafety requirements. Detailed analysis of in vitro neutralization activity requires more sophisticated methods that have to be performed under higher biosafety levels.