An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 in human airway epithelial cell cultures and multiple coronaviruses in mice.

Coronaviruses (CoVs) traffic frequently between species resulting in novel disease outbreaks, most recently exemplified by the newly emerged SARS-CoV-2, the causative agent of COVID-19. Here, we show that the ribonucleoside analog ß-d-N4-hydroxycytidine (NHC; EIDD-1931) has broad-spectrum antiviral activity against SARS-CoV-2, MERS-CoV, SARS-CoV, and related zoonotic group 2b or 2c bat-CoVs, as well as increased potency against a CoV bearing resistance mutations to the nucleoside analog inhibitor remdesivir. In mice infected with SARS-CoV or MERS-CoV, both prophylactic and therapeutic administration of EIDD-2801, an orally bioavailable NHC prodrug (ß-d-N4-hydroxycytidine-5'-isopropyl ester), improved pulmonary function and reduced virus titer and body weight loss. Decreased MERS-CoV yields in vitro and in vivo were associated with increased transition mutation frequency in viral, but not host cell RNA, supporting a mechanism of lethal mutagenesis in CoV. The potency of NHC/EIDD-2801 against multiple CoVs and oral bioavailability highlights its potential utility as an effective antiviral against SARS-CoV-2 and other future zoonotic CoVs.

Antibody-mediated synergy and interference in the neutralization of SARS-CoV at an epitope cluster on the spike protein.

Incomplete neutralization of virus, especially when it occurs in the presence of excess neutralizing antibody, represents a biological phenomenon that impacts greatly on antibody-mediated immune prophylaxis of viral infection and on successful vaccine design. To understand the mechanism by which a virus escapes from antibody-mediated neutralization, we have investigated the interactions of non-neutralizing and neutralizing antibodies at an epitope cluster on the spike protein of severe acute respiratory syndrome coronavirus (SARS-CoV). The epitope cluster was mapped at the C-terminus of the spike protein; it consists of structurally intertwined epitopes recognized by two neutralizing monoclonal antibodies (mAbs), 341C and 540C, and a non-neutralizing mAb, 240C. While mAb 341C binds to a mostly linear epitope composed of residues (507)PAT(509) and V(349), mAb 240C binds to an epitope that partially overlaps the former by at least two residues (P(507) and A(508)). The epitope corresponding to mAb 540C is a conformational one, involving residues L(504) and N(505). In neutralization assays, non-neutralizing 240C disrupted virus neutralization by mAb 341C and/or mAb 540C, whereas a combination of mAbs 341C and 540C blocked virus infectivity synergistically. These findings indicate that the epitope cluster on the spike protein may serve as an evolutionarily conserved platform at which a dynamic interplay between neutralizing and non-neutralizing antibodies occurs, thereby determining the outcome of SARS-CoV infection.

Inhibition of measles virus infections in cell cultures by peptide-conjugated morpholino oligomers.

Measles virus (MeV) is a highly contagious human pathogen. Despite the success of measles vaccination programs, measles is still responsible for an estimated 245,000 deaths each year. There are currently no antiviral compounds available for the treatment of measles. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) are antisense compounds that enter cells readily and can interfere with mRNA function by steric blocking. A panel of PPMO was designed to target various sequences of MeV RNA that are known to be important for viral replication. Five PPMO, targeting MeV genomic RNA or mRNA, inhibited the replication of MeV, in a dose-responsive and sequence-specific manner in cultured cells. One of the highly active PPMO (PPMO 454), targeting a conserved sequence in the translation start site of the mRNA coding for the nucleocapsid protein, inhibited multiple genotypes of MeV. This report provides evidence that PPMO treatment represents a promising approach for developing antiviral agents against measles and other paramyxoviruses.

Induction of neutralising antibodies and cellular immune responses against SARS coronavirus by recombinant measles viruses.

Live attenuated recombinant measles viruses (rMV) expressing a codon-optimised spike glycoprotein (S) or nucleocapsid protein (N) of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) were generated (rMV-S and rMV-N). Both recombinant viruses stably expressed the corresponding SARS-CoV proteins, grew to similar end titres as the parental strain and induced high antibody titres against MV and the vectored SARS-CoV antigens (S and N) in transgenic mice susceptible to measles infection. The antibodies induced by rMV-S had a high neutralising effect on SARS-CoV as well as on MV. Moreover, significant N-specific cellular immune responses were measured by IFN-gamma ELISPOT assays. The pre-existence of anti-MV antibodies induced by the initial immunisation dose did not inhibit boost of anti-S and anti-N antibodies. Immunisations comprising a mixture of rMV-S and rMV-N induced immune responses similar in magnitude to that of vaccine components administered separately. These data support the suitability of MV as a bivalent candidate vaccine vector against MV and emerging viruses such as SARS-CoV.

Monoclonal antibodies to SARS-associated coronavirus (SARS-CoV): identification of neutralizing and antibodies reactive to S, N, M and E viral proteins.

Monoclonal antibodies (Mabs) against the Urbani strain of the SARS-associated coronavirus (SARS-CoV) were developed and characterized for reactivity to SARS-CoV and SARS-CoV S, N, M, and E proteins using enzyme-linked immunoabsorbent (ELISA), radioimmunoprecipitation, immunofluorescence, Western Blot and microneutralization assays. Twenty-six mAbs were reactive to SARS-CoV by ELISA, and nine were chosen for detailed characterization. Five mAbs reacted against the S protein, two against the M protein, and one each against the N and E proteins. Two of five S protein mAbs neutralized SARS-CoV infection of Vero E6 cells and reacted to an epitope within amino acids 490-510 in the S protein. While two of the three non-neutralizing antibodies recognized at second epitope within amino acids 270-350. The mAbs characterized should prove useful for developing SARS-CoV diagnostic assays and for studying the biology of infection and pathogenesis of disease.

SARS-coronavirus replication in human peripheral monocytes/macrophages.

A novel coronavirus (CoV) has been described in association with cases of severe acute respiratory syndrome (SARS). The virus, SARS-CoV, differs from the previously described human coronaviruses, 229E and OC43. 229E was previously shown to productively infect human monocytes/macrophages, whereas OC43 poorly infected the cells. In this study, we examined whether SARS-CoV could productively infect purified monocytes/macrophages (PM) derived from human donor cells. Unlike 229E-infected cells, which produced viral titers of 10(3.5) to 10(6)TCID50/ml, SARS-CoV replicated poorly in PM, producing titers of 10(1.75) to 10(2)TCID50/ml. This finding was similar to results reported for OC43-infected cells, with titers ranging from 10(1.2) to 10(2.7)TCID50/ml. Of interest, SARS-CoV proteins were detected only in PM that did not produce significant amounts of interferon (IFN)-alpha, and in one such case, preliminary electron microscope studies demonstrated that SARS-CoV-like particles could enter the cells, possibly via phagocytosis. These results suggest that SARS-CoV, like human CoV OC43, poorly infects human PM, and production of IFN-alpha by these cells further limits the infection. Given the importance of monocytes/macrophages to the immune response, it is possible that their infection by SARS-CoV and alteration of this infection by IFN-alpha may be important to the course of the infection in humans.

Effects of a SARS-associated coronavirus vaccine in monkeys.

The causative agent of severe acute respiratory syndrome (SARS) has been identified as a new type of coronavirus. Here, we have investigated the ability of adenoviral delivery of codon-optimised SARS-CoV strain Urbani structural antigens spike protein S1 fragment, membrane protein, and nucleocapsid protein to induce virus-specific broad immunity in rhesus macaques. We immunised rhesus macaques intramuscularly with a combination of the three Ad5-SARS-CoV vectors or a control vector and gave a booster vaccination on day 28. The vaccinated animals all had antibody responses against spike protein S1 fragment and T-cell responses against the nucleocapsid protein. All vaccinated animals showed strong neutralising antibody responses to SARS-CoV infection in vitro. These results show that an adenoviral-based vaccine can induce strong SARS-CoV-specific immune responses in the monkey, and hold promise for development of a protective vaccine against the SARS causal agent.

Characterization of a novel coronavirus associated with severe acute respiratory syndrome.

In March 2003, a novel coronavirus (SARS-CoV) was discovered in association with cases of severe acute respiratory syndrome (SARS). The sequence of the complete genome of SARS-CoV was determined, and the initial characterization of the viral genome is presented in this report. The genome of SARS-CoV is 29,727 nucleotides in length and has 11 open reading frames, and its genome organization is similar to that of other coronaviruses. Phylogenetic analyses and sequence comparisons showed that SARS-CoV is not closely related to any of the previously characterized coronaviruses.

Functional properties of the fusion and attachment glycoproteins of Nipah virus.

Nipah virus (NV) and Hendra virus (HV) are recently emergent, related viruses that can cause severe disease in humans and animals. The goal of this study was to investigate the immunogenic and functional properties of the fusion (F) and attachment (G) glycoproteins of NV. Vaccination of mice with recombinant vaccinia viruses (rVVs) expressing either the F (rVV/NV-F) or G (rVV/NV-G) proteins of NV induced neutralizing antibody responses to NV, with higher titers produced after vaccination with rVV/NV-G. When the homologous pairs of F and G proteins from either HV or NV were coexpressed in a transient expression system, fusion was detected in less than 12 h. An equivalent amount of fusion was observed when the heterologous pairs of F and G proteins from HV and NV were coexpressed. Membrane fusion was inhibited by antiserum from mice vaccinated with rVV/NV-G and rVV/NV-F. Therefore, as with other paramyxoviruses, the membrane glycoproteins of NV are the targets of neutralizing antibodies and membrane fusion mediated by NV requires the presence of both the F and the G proteins. Data from these biological assays support the taxonomic grouping of both HV and NV in the new genus, Henipavirus, within the family Paramyxoviridae.