COVA1-18 neutralizing antibody protects against SARS-CoV-2 in three preclinical models.

One year into the Coronavirus Disease 2019 (COVID-19) pandemic caused by Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), effective treatments are still needed 1-3 . Monoclonal antibodies, given alone or as part of a therapeutic cocktail, have shown promising results in patients, raising the hope that they could play an important role in preventing clinical deterioration in severely ill or in exposed, high risk individuals 4-6 . Here, we evaluated the prophylactic and therapeutic effect of COVA1-18 in vivo , a neutralizing antibody isolated from a convalescent patient 7 and highly potent against the B.1.1.7. isolate 8,9 . In both prophylactic and therapeutic settings, SARS-CoV-2 remained undetectable in the lungs of COVA1-18 treated hACE2 mice. Therapeutic treatment also caused a dramatic reduction in viral loads in the lungs of Syrian hamsters. When administered at 10 mg kg - 1 one day prior to a high dose SARS-CoV-2 challenge in cynomolgus macaques, COVA1-18 had a very strong antiviral activity in the upper respiratory compartments with an estimated reduction in viral infectivity of more than 95%, and prevented lymphopenia and extensive lung lesions. Modelling and experimental findings demonstrate that COVA1-18 has a strong antiviral activity in three different preclinical models and could be a valuable candidate for further clinical evaluation.

Mapping of two dominant sites of VP35 of Marburg virus.

Five types of anti-VP35 monoclonal antibodies (MAbs), four immune sera against Marburg virus (MBGV), and 11 overlapping recombinant VP35 fragments were used to map the epitopes for VP35 of MBGV. The purified full-size recombinant VP35 was highly immunogenic and retained the B-cell epitopes that were identical to those of the viral VP35. Two major sites on VP35 and a set of truncated VP35 fragments were found by use of an enzyme immunoassay and immunoblot. Site I was located in a region between amino acids 1 and 174 of the VP35 sequence, and only polyclonal antibodies (PAbs) against MBGV recognized epitopes at this site. Site II was mapped by use of anti-VP35 MAbs to the region between amino acid residues 167 and 278 of VP35. Amino acids 252-278 of VP35 might be involved in the formation of the epitopes for MAbs. B-cell epitopes were not found on the C-terminus of VP35 by use of PAbs or MAbs.

Granulocyte-macrophage colony-stimulating factor expressed by recombinant respiratory syncytial virus attenuates viral replication and increases the level of pulmonary antigen-presenting cells.

An obstacle to developing a vaccine against human respiratory syncytial virus (RSV) is that natural infection typically does not confer solid immunity to reinfection. To investigate methods to augment the immune response, recombinant RSV (rRSV) was constructed that expresses murine granulocyte-macrophage colony-stimulating factor (mGM-CSF) from a transcription cassette inserted into the G-F intergenic region. Replication of rRSV/mGM-CSF in the upper and lower respiratory tracts of BALB/c mice was reduced 23- to 74- and 5- to 588-fold, respectively, compared to that of the parental rRSV. Despite this strong attenuation of replication, the level of RSV-specific serum antibodies induced by rRSV/mGM-CSF was comparable to, or marginally higher than, that of the parental rRSV. The induction of RSV-specific CD8(+) cytotoxic T cells was moderately reduced during the initial infection, which might be a consequence of reduced antigen expression. Mice infected with rRSV/mGM-CSF had elevated levels of pulmonary mRNA for gamma interferon (IFN-gamma) and interleukin 12 (IL-12) p40 compared to animals infected by wild-type rRSV. Elevated synthesis of IFN-gamma could account for the restriction of RSV replication, as was observed previously with an IFN-gamma-expressing rRSV. The accumulation of total pulmonary mononuclear cells and total CD4(+) T lymphocytes was accelerated in animals infected with rRSV/mGM-CSF compared to that in animals infected with the control virus, and the level of IFN-gamma-positive or IL-4-positive pulmonary CD4(+) cells was elevated approximately twofold. The number of pulmonary lymphoid and myeloid dendritic cells and macrophages was increased up to fourfold in mice infected with rRSV/mGM-CSF compared to those infected with the parental rRSV, and the mean expression of major histocompatibility complex class II molecules, a marker of activation, was significantly increased in the two subsets of dendritic cells. Enhanced antigen presentation likely accounts for the maintenance of a strong antibody response despite reduced viral replication and would be a desirable property for a live attenuated rRSV vaccine.

Respiratory syncytial virus can tolerate an intergenic sequence of at least 160 nucleotides with little effect on transcription or replication in vitro and in vivo.

The intergenic sequences (IGS) between the first nine genes of human respiratory syncytial virus (RSV) vary in length from 1 to 56 nucleotides and lack apparent conserved sequence motifs. To investigate their influence on sequential transcription and viral growth, recombinant RSV strain A2, from which the SH gene had been deleted to facilitate manipulation, was further modified to contain an M-G IGS of 16, 30, 44, 58, 65, 72, 86, 100, 120, 140, or 160 nucleotides. All of the viruses were viable. For viruses with an M-G IGS of 100 nucleotides or more, plaque size decreased with increasing IGS length. In this same length range, increasing IGS length was associated with modest attenuation during single-step, but not multistep, growth in HEp-2 cells. Surprisingly, Northern blot analysis of the accumulation of six different mRNAs indicated that there was little or no change in transcription with increasing IGS length. Thus, the RSV polymerase apparently can readily cross IGS of various lengths, including unnaturally long ones, with little or no effect on the efficiency of termination and reinitiation. This finding supports the view that the IGS do not have much effect on sequential transcription and provides evidence from infectious virus that IGS length is not an important regulatory feature. To evaluate replication in vivo, BALB/c mice were infected intranasally with RSV containing an M-G IGS of 65, 140, or 160 nucleotides. Replication of the latter two viruses was decreased up to 5- and 25-fold in the upper and lower respiratory tracts, respectively, on day 3 following infection. However, the level of replication at both sites on days 4 and 5 was very similar to that of the virus with an IGS of 65 nucleotides. Thus, the modest attenuation in vivo associated with the longer IGS was additive to that conferred by deletion of the SH gene and might be useful to incrementally increase the level of attenuation of a live-attenuated vaccine virus.

Effect of coexpression of interleukin-2 by recombinant respiratory syncytial virus on virus replication, immunogenicity, and production of other cytokines.

We constructed rRSV/mIL-2, a recombinant respiratory syncytial virus (rRSV) containing the coding sequence of murine interleukin-2 (mIL-2) in a transcription cassette inserted into the G-F intergenic region. The recovered virus (rRSV/mIL-2) expressed high levels (up to 2.8 microg/ml) of mIL-2 in cell culture. Replication of rRSV/mIL-2 in vitro was reduced up to 13.6-fold from that of wild-type (wt) rRSV, an effect that was due to the presence of the foreign insert but was not specific to mIL-2. Replication of the rRSV/mIL-2 virus in the upper and lower respiratory tracts of BALB/c mice was reduced up to 6.3-fold, an effect that was specific to mIL-2. The antibody response, including the levels of RSV-specific serum immunoglobulin G1 (IgG1), IgG2a, IgA, and total IgG, and the level of protective efficacy against wt RSV challenge were not significantly different from those of wt rRSV. Analysis of total pulmonary cytokine mRNA isolated 1 and 4 days following infection with rRSV/mIL-2 revealed elevated levels of mRNA for IL-2, gamma interferon (IFN-gamma), IL-4, IL-5, IL-6, IL-10, IL-13, and IL-12 p40 compared to those for wt rRSV. Flow cytometry of total pulmonary mononuclear cells isolated 10 days following infection with rRSV/mIL-2 revealed increased levels of CD4(+) T lymphocytes expressing either IFN-gamma or IL-4 compared to those of wt rRSV. These elevations in cytokine mRNA or cytokine-expressing CD4(+) cells relative to those of wt rRSV-primed animals were not observed following challenge with wt RSV on day 28. Thus, the expression of mIL-2 by rRSV was associated with a modest attenuation of virus growth in vivo, induction of serum antibodies at levels comparable to that of wt rRSV, and transient increases in both the Th1 and Th2 CD4(+) lymphocytes and cytokine mRNAs compared to those of wt rRSV.

Rational design of live-attenuated recombinant vaccine virus for human respiratory syncytial virus by reverse genetics.

RSV is a major cause of pediatric respiratory tract disease worldwide, but a vaccine is not yet available. It is now possible to prepare live infectious RSV completely from cDNA. This provides a method for introducing defined mutations into infectious virus, making possible the rational design of a live-attenuated vaccine virus for intranasal administration. This is particularly important for RSV, for which achieving the appropriate balance between attenuation and immunogenicity by conventional methods has proven elusive. We took advantage of the existence of a panel of biologically derived vaccine candidate viruses that were incompletely attenuated but well characterized biologically. The mutations in these viruses were identified by sequence analysis and characterized by insertion into recombinant virus, thereby providing a menu of known attenuating mutations. These included a series of amino acid point mutations, mostly in the L polymerase, and a nucleotide substitution in a transcription gene-start signal, a cis-acting RNA element. The second source of mutations was from experimental mutational analysis of recombinant virus and involves deletion of the NS1, NS2, or SH gene. We have reconstructed a previously tested, biologically derived attenuated virus, cpts248/404, in recombinant form and are now proceeding to introduce additional mutations from the menu to achieve stepwise increases in attenuation. The ability to modify the attenuation phenotype incrementally in a directed manner should result in an appropriate vaccine virus.

Interferon gamma expressed by a recombinant respiratory syncytial virus attenuates virus replication in mice without compromising immunogenicity.

Interferon gamma (IFN-gamma) has pleiotropic biological effects, including intrinsic antiviral activity as well as stimulation and regulation of immune responses. An infectious recombinant human respiratory syncytial virus (rRSV/mIFN-gamma) was constructed that encodes murine (m) IFN-gamma as a separate gene inserted into the G-F intergenic region. Cultured cells infected with rRSV/mIFN-gamma secreted 22 microg mIFN-gamma per 10(6) cells. The replication of rRSV/mIFN-gamma, but not that of a control chimeric rRSV containing the chloramphenicol acetyl transferase (CAT) gene as an additional gene, was 63- and 20-fold lower than that of wild-type (wt) RSV in the upper and lower respiratory tract, respectively, of mice. Thus, the attenuation of rRSV/mIFN-gamma in vivo could be attributed to the activity of mIFN-gamma and not to the presence of the additional gene per se. The mice were completely resistant to subsequent challenge with wt RSV. Despite its growth restriction, infection of mice with rRSV/mIFN-gamma induced a level of RSV-specific antibodies that, on day 56, was comparable to or greater than that induced by infection with wt RSV. Mice infected with rRSV/mIFN-gamma developed a high level of IFN-gamma mRNA and an increased amount of interleukin 12 p40 mRNA in their lungs, whereas other cytokine mRNAs tested were unchanged compared with those induced by wt RSV. Because attenuation of RSV typically is accompanied by a reduction in immunogenicity, expression of IFN-gamma by an rRSV represents a method of attenuation in which immunogenicity can be maintained rather than be reduced.

Recombinant respiratory syncytial virus bearing a deletion of either the NS2 or SH gene is attenuated in chimpanzees.

The NS2 and SH genes of respiratory syncytial virus (RSV) have been separately deleted from a recombinant wild-type RSV strain, A2 (M. N. Teng and P. L. Collins, J. Virol. 73:466-473, 1998; A. Bukreyev et al., J. Virol. 71:8973-8982, 1997; and this study). The resulting viruses, designated rA2DeltaNS2 and rA2DeltaSH, were administered to chimpanzees to evaluate their levels of attenuation and immunogenicity. Recombinant virus rA2DeltaNS2 replicated to moderate levels in the upper respiratory tract, was highly attenuated in the lower respiratory tract, and induced significant resistance to challenge with wild-type RSV. The replication of rA2DeltaSH virus was only moderately reduced in the lower, but not the upper, respiratory tract. However, chimpanzees infected with either virus developed significantly less rhinorrhea than those infected with wild-type RSV. These findings demonstrate that a recombinant RSV mutant lacking either the NS2 or SH gene is attenuated and indicate that these deletions may be useful as attenuating mutations in new, live recombinant RSV vaccine candidates for both pediatric and elderly populations. The DeltaSH mutation was incorporated into a recombinant form of the cpts248/404 vaccine candidate, was evaluated for safety in seronegative chimpanzees, and can now be evaluated as a vaccine for humans.

Characteristics of Filoviridae: Marburg and Ebola viruses.

Filoviruses are enveloped, nonsegmented negative-stranded RNA viruses. The two species, Marburg and Ebola virus, are serologically, biochemically, and genetically distinct. Marburg virus was first isolated during an outbreak in Europe in 1967, and Ebola virus emerged in 1976 as the causative agent of two simultaneous outbreaks in southern Sudan and northern Zaire. Although the main route of infection is known to be person-to-person transmission by intimate contact, the natural reservoir for filoviruses still remains a mystery.

Recombinant respiratory syncytial virus from which the entire SH gene has been deleted grows efficiently in cell culture and exhibits site-specific attenuation in the respiratory tract of the mouse.

The small hydrophobic protein SH of human respiratory syncytial virus (RSV) is a short transmembrane surface protein of unknown function. A full-length cDNA of RSV strain A2 (subgroup A) antigenomic RNA was modified such that the entire SH gene, including the transcription signals and the complete mRNA-encoding sequence, was deleted and replaced by a synthetic intergenic region. This reduced the length of the antigenome by 398 nucleotides and ablated expression of 1 of the 10 RSV mRNAs. Recombinant virus containing this engineered deletion was recovered, and the absence of the SH gene was confirmed by reverse transcription in conjunction with PCR. Northern blot analysis of intracellular RNAs and gel electrophoresis of labeled intracellular proteins confirmed the lack of expression of the SH mRNA and protein. The absence of the SH gene did not noticeably affect RNA replication, but two effects on transcription were noted. First, synthesis of the G, F, and M2 mRNAs was increased, presumably due to their being one position closer to the promoter in the gene order. Second, transcription of genes downstream of the engineered site exhibited a steeper gradient of polarity. On monolayers of HEp-2 cells, the SH-minus virus produced syncytia which were at least equivalent in size to those of the wild type and produced plaques which were 70% larger. Furthermore, the SH-minus virus grew somewhat better (up to 12.6-fold) than wild-type recombinant RSV in certain cell lines. While the function of the SH protein remains to be determined, it seems to be completely dispensable for growth in tissue culture and fusion function. When inoculated intranasally into mice, the SH-minus virus resembled the wild-type recombinant virus in its efficiency of replication in the lungs, whereas it replicated 10-fold less efficiently in the upper respiratory tract. In mice, the SH-minus and wild-type recombinant viruses were similarly immunogenic and effective in inducing resistance to virus challenge.

Recovery of infectious respiratory syncytial virus expressing an additional, foreign gene.

A previous report described the recovery from cDNA of infectious recombinant respiratory syncytial virus (RSV) strain A2 (P. L. Collins, M. G. Hill, E. Camargo, H. Grosfeld, R. M. Chanock, and B. R. Murphy, Proc. Natl. Acad. Sci. USA, 92:11563-11567, 1995). Here, the system was used to construct recombinant RSV containing an additional gene encoding chloramphenicol acetyltransferase (CAT). The CAT coding sequence was flanked by RSV-specific gene-start and gene-end motifs, the transcription signals for the viral RNA-dependent RNA polymerase. The RSV-CAT chimeric transcription cassette was inserted into the region between the G and F genes of the complete cDNA-encoded positive-sense RSV antigenome, and infectious CAT-expressing recombinant RSV was recovered. Transcription of the inserted gene into the predicted subgenomic polyadenylated mRNA was demonstrated by Northern (RNA) blot hybridization analysis, and the encoded protein was detected by enzyme assay and by radioimmunoprecipitation. Quantitation of intracellular CAT, SH, G, and F mRNAs showed that the CAT mRNA was efficiently expressed and that the levels of the G and F mRNAs (which represent the genes on either side of the inserted CAT gene) were comparable to those expressed by a wild-type recombinant RSV. Consistent with this finding, the CAT-containing and wild-type viruses were very similar with regard to the levels of synthesis of the major viral proteins. Each of 25 RSV isolates obtained by plaque purification following eight serial passages expressed CAT, showing that the foreign gene was faithfully maintained in functional form. Analysis by reverse transcription and PCR did not reveal evidence of deletion of the foreign sequence. This finding demonstrated that the RSV genome can accept and maintain an increase in length of 762 nucleotides of foreign sequence and can be engineered to encode an additional, 11th mRNA. The presence of the additional gene resulted in a 10% decrease in plaque diameter and was associated with delay in virus growth and 20-fold decrease in virus yield in vitro. Thus, introduction of an additional gene into the RSV genome might represent a method of attenuation. The ability to express foreign genes by recombinant RSV has implications for basic studies as well as for the development of live recombinant vaccines.

Complete nucleotide sequences of Marburg virus genes 5 and 6 encoding VP30 and VP24 proteins.

Nucleotide sequences of the genes 5 and 6 of the Marburg virus, Popp strain, were determined. ORFs encoding polypeptides VP30 (281 a.a., MW 32,640) and VP24 (253 a.a., MW 28,621) were found. The putative transcription start and stop signals for viral RNA-dependent RNA polymerase were revealed for both genes. Overlapping of genes 5 and 6 was shown. The deduced amino acid sequences of VP30 and VP24 proteins displayed significant homology with the analogous proteins of another filovirus, the Ebola virus (33% and 37%, respectively). The VP24 appeared to have a hydrophobic amino acid composition; content of hydrophobic amino acids was 40.7%. Model of VP24 location in the virion was suggested.

The complete nucleotide sequence of the Popp (1967) strain of Marburg virus: a comparison with the Musoke (1980) strain.

The nucleotide sequence of genomic RNA of Marburg virus strain Popp was determined. Strain Popp was isolated in 1967 during the first filoviral outbreak. The virus was purified from blood of infected guinea pigs in which it had been maintained. The length of the determined sequence was 19112 nucleotides. Amino acid sequences of seven known virion proteins were deduced. Nucleotide and amino acid sequences were compared with those of strain Musoke of Marburg virus isolated in 1980 in Kenya and purified from Vero cells. Homology between nucleotide sequences of two strains was 93.9%. Comparisons revealed conserved and variable regions of the nucleotide and amino acid sequences. The GP, the envelope protein of the virion, was found to be the most variable protein. The greatest differences in the protein were located in the supposedly external part of the molecule. Amino acid substitutions in the L protein, the main component of viral RNA-dependent RNA polymerase, were also distributed extremely non-randomly. It was shown that the non-coding regions of the genome were more variable than the coding ones; 37.6% of nucleotide differences corresponded to the former. 72.6% of nucleotide substitutions located in the coding regions were found to be at the third codon position.

The GP-protein of Marburg virus contains the region similar to the 'immunosuppressive domain' of oncogenic retrovirus P15E proteins.

cDNA was synthesized and cloned on the template of the genomic RNA of Marburg virus (strain Popp). Recombinant plasmids with specific cDNA inserts were selected and sequenced. The length of the open reading frame encoding the GP-protein is 681 amino acids. GP-protein is proposed to be an integral membrane protein. Computer-assisted comparison of the deduced amino acid sequence with those of different viruses revealed significant homology with the GP-protein of Ebola virus and with the 'immunosuppressive domain' of the P15E envelope proteins of some oncogenic retroviruses.

The VP35 and VP40 proteins of filoviruses. Homology between Marburg and Ebola viruses.

The fragments of genomic RNA sequences of Marburg (MBG) and Ebola (EBO) viruses are reported. These fragments were found to encode the VP35 and VP40 proteins. The canonic sequences were revealed before and after each open reading frame. It is suggested that these sequences are mRNA extremities and at the same time the regulatory elements for mRNA transcription. Homology between the MBG and EBO proteins was discovered.