The SARS-CoV-2 Y453F mink variant displays a pronounced increase in ACE-2 affinity but does not challenge antibody neutralization

Transmission of Severe Acute Respiratory Syndrome Coronavirus 2 from humans to animals has been reported for many domesticated species, including farmed minks. The identification of novel spike gene mutations appearing in minks has raised major concerns about potential immune evasion and challenges for the global vaccine strategy. One genetic variant, known as “cluster five,” arose among farmed minks in Denmark and resulted in a complete shutdown of the world’s largest mink production. However, the functional properties of this new variant are not established. Here we present functional data on the cluster-five variant, which contains a mutation resulting in a Y453F residue change in the receptor-binding domain (RBD) of the spike protein. Using an ELISA-based angiotensin-converting enzyme-2/RBD inhibition assay, we show that the Y453F variant does not decrease established humoral immunity from previously infected individuals or affect the neutralizing antibody response in a vaccine mouse model based on the original Wuhan strain RBD or spike as antigens. However, biolayer interferometry analysis demonstrates that it binds the human angiotensin-converting enzyme-2 receptor with a 4-fold higher affinity than the original strain, suggesting an enhanced transmission capacity and a possible challenge for viral control. These results also indicate that the rise in the frequency of the cluster-five variant in mink farms might be a result of the fitness advantage conferred by the receptor adaptation rather than evading immune responses. Transmission of Severe Acute Respiratory Syndrome Coronavirus 2 from humans to animals has been reported for many domesticated species, including farmed minks. The identification of novel spike gene mutations appearing in minks has raised major concerns about potential immune evasion and challenges for the global vaccine strategy. One genetic variant, known as “cluster five,” arose among farmed minks in Denmark and resulted in a complete shutdown of the world’s largest mink production. However, the functional properties of this new variant are not established. Here we present functional data on the cluster-five variant, which contains a mutation resulting in a Y453F residue change in the receptor-binding domain (RBD) of the spike protein. Using an ELISA-based angiotensin-converting enzyme-2/RBD inhibition assay, we show that the Y453F variant does not decrease established humoral immunity from previously infected individuals or affect the neutralizing antibody response in a vaccine mouse model based on the original Wuhan strain RBD or spike as antigens. However, biolayer interferometry analysis demonstrates that it binds the human angiotensin-converting enzyme-2 receptor with a 4-fold higher affinity than the original strain, suggesting an enhanced transmission capacity and a possible challenge for viral control. These results also indicate that the rise in the frequency of the cluster-five variant in mink farms might be a result of the fitness advantage conferred by the receptor adaptation rather than evading immune responses. Reply to Lassaunière: On the functional characterization of the Y453F RBD variant found in cluster 5 SARS-CoV-2Journal of Biological ChemistryVol. 297Issue 5PreviewWe appreciate the opportunity to comment on the letter from Dr Lassaunière. Full-Text PDF Open AccessSARS-CoV-2 Y453F is not the “cluster 5” variantJournal of Biological ChemistryVol. 297Issue 5PreviewI read with interest the recent publication by Bayarri-Olmos et al. (1) and would like to express some concerns regarding the study. Full-Text PDF Open Access Experimental infection models have shown that Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is capable of infecting a wide range of animal species, such as cats, dogs, ferrets, and hamsters (1Shi J. Wen Z. Zhong G. Yang H. Wang C. Huang B. Liu R. He X. Shuai L. Sun Z. Zhao Y. Liu P. Liang L. Cui P. Wang J. et al.Susceptibility of ferrets, cats, dogs, and other domesticated animals to SARS-coronavirus 2.Science. 2020; 368: 1016-1020Crossref PubMed Scopus (1182) Google Scholar, 2Halfmann P.J. Hatta M. Chiba S. Maemura T. Fan S. Takeda M. Kinoshita N. Hattori S. Sakai-Tagawa Y. Iwatsuki-Horimoto K. Imai M. Kawaoka Y. Transmission of SARS-CoV-2 in domestic cats.N. Engl. J. Med. 2020; 383: 592-594Crossref PubMed Scopus (331) Google Scholar, 3Sia S.F. Yan L.-M. Chin A.W.H. Fung K. Choy K.-T. Wong A.Y.L. Kaewpreedee P. Perera R.A.P.M. Poon L.L.M. Nicholls J.M. Peiris M. Yen H.-L. Pathogenesis and transmission of SARS-CoV-2 in golden hamsters.Nature. 2020; 583: 834-838Crossref PubMed Scopus (827) Google Scholar), and natural reverse-zoonotic transmission to animals has been documented in farmed mink (4Oreshkova N. Molenaar R.J. Vreman S. Harders F. Oude Munnink B.B. Hakze-van der Honing R.W. Gerhards N. Tolsma P. Bouwstra R. Sikkema R.S. Tacken M.G. de Rooij M.M. Weesendorp E. Engelsma M.Y. Bruschke C.J. et al.SARS-CoV-2 infection in farmed minks, The Netherlands, April and May 2020.Euro Surveill. 2020; 25: 2001005Crossref PubMed Scopus (426) Google Scholar), dogs (5Sit T.H.C. Brackman C.J. Ip S.M. Tam K.W.S. Law P.Y.T. To E.M.W. Yu V.Y.T. Sims L.D. Tsang D.N.C. Chu D.K.W. Perera R.A.P.M. Poon L.L.M. Peiris M. Infection of dogs with SARS-CoV-2.Nature. 2020; 586: 776-778Crossref PubMed Scopus (451) Google Scholar, 6Patterson E.I. Elia G. Grassi A. Giordano A. Desario C. Medardo M. Smith S.L. Anderson E.R. Prince T. Patterson G.T. Lorusso E. Lucente M.S. Lanave G. Lauzi S. Bonfanti U. et al.Evidence of exposure to SARS-CoV-2 in cats and dogs from households in Italy.Nat. Commun. 2020; 11: 6231Crossref PubMed Scopus (205) Google Scholar), and felines (6Patterson E.I. Elia G. Grassi A. Giordano A. Desario C. Medardo M. Smith S.L. Anderson E.R. Prince T. Patterson G.T. Lorusso E. Lucente M.S. Lanave G. Lauzi S. Bonfanti U. et al.Evidence of exposure to SARS-CoV-2 in cats and dogs from households in Italy.Nat. Commun. 2020; 11: 6231Crossref PubMed Scopus (205) Google Scholar, 7Newman A. Smith D. Ghai R.R. Wallace R.M. Torchetti M.K. Loiacono C. Murrell L.S. Carpenter A. Moroff S. Rooney J.A. Barton Behravesh C. First reported cases of SARS-CoV-2 infection in companion animals - New York, March-April 2020.MMWR. Morb. Mortal. Wkly. Rep. 2020; 69: 710-713Crossref PubMed Google Scholar, 8Yoo H.S. Yoo D. COVID-19 and veterinarians for one health, zoonotic- and reverse-zoonotic transmissions.J. Vet. Sci. 2020; 21: e51Crossref PubMed Scopus (47) Google Scholar). Several variants affecting the spike protein have been documented since the early reports in the spring of 2020 of SARS-CoV-2 transmission from humans to minks (4Oreshkova N. Molenaar R.J. Vreman S. Harders F. Oude Munnink B.B. Hakze-van der Honing R.W. Gerhards N. Tolsma P. Bouwstra R. Sikkema R.S. Tacken M.G. de Rooij M.M. Weesendorp E. Engelsma M.Y. Bruschke C.J. et al.SARS-CoV-2 infection in farmed minks, The Netherlands, April and May 2020.Euro Surveill. 2020; 25: 2001005Crossref PubMed Scopus (426) Google Scholar, 9Molenaar R.J. Vreman S. Hakze-van der Honing R.W. Zwart R. de Rond J. Weesendorp E. Smit L.A.M. Koopmans M. Bouwstra R. Stegeman A. van der Poel W.H.M. Clinical and pathological findings in SARS-CoV-2 disease outbreaks in farmed mink (Neovison vison).Vet. Pathol. 2020; 57: 653-657Crossref PubMed Scopus (108) Google Scholar, 10Oude Munnink B.B. Sikkema R.S. Nieuwenhuijse D.F. Molenaar R.J. Munger E. Molenkamp R. van der Spek A. Tolsma P. Rietveld A. Brouwer M. Bouwmeester-Vincken N. Harders F. der Honing R.H. Wegdam-Blans M.C.A. Bouwstra R.J. et al.Jumping back and forth: Anthropozoonotic and zoonotic transmission of SARS-CoV-2 on mink farms.bioRxiv. 2021; : 1-35https://doi.org/10.1101/2020.09.01.277152Crossref Scopus (0) Google Scholar). The spike protein mediates the binding of SARS-CoV-2 to the human angiotensin-converting enzyme-2 (ACE-2) receptor (11Walls A.C. Park Y.-J.J. Tortorici M.A. Wall A. McGuire A.T. Veesler D. Structure, function, and antigenicity of the SARS-CoV-2 spike Glycoprotein.Cell. 2020; 181: 281-292.e6Abstract Full Text Full Text PDF PubMed Scopus (5486) Google Scholar, 12Hoffmann M. Kleine-Weber H. Schroeder S. Krüger N. Herrler T. Erichsen S. Schiergens T.S. Herrler G. Wu N.-H.H. Nitsche A. Müller M.A. Drosten C. Pöhlmann S. SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor.Cell. 2020; 181: 271-280.e8Abstract Full Text Full Text PDF PubMed Scopus (12453) Google Scholar) and strongly influences the viral infectivity and transmissibility. Moreover, the spike protein is the main target for the host's protective antibody response (11Walls A.C. Park Y.-J.J. Tortorici M.A. Wall A. McGuire A.T. Veesler D. Structure, function, and antigenicity of the SARS-CoV-2 spike Glycoprotein.Cell. 2020; 181: 281-292.e6Abstract Full Text Full Text PDF PubMed Scopus (5486) Google Scholar, 13He Y. Zhou Y. Wu H. Luo B. Chen J. Li W. Jiang S. Identification of immunodominant sites on the spike protein of severe acute respiratory syndrome (SARS) coronavirus: Implication for developing SARS diagnostics and vaccines.J. Immunol. 2004; 173: 4050-4057Crossref PubMed Scopus (128) Google Scholar, 14Du L. He Y. Zhou Y. Liu S. Zheng B.J. Jiang S. The spike protein of SARS-CoV - a target for vaccine and therapeutic development.Nat. Rev. Microbiol. 2009; 7: 226-236Crossref PubMed Scopus (1166) Google Scholar). Mutations arising through antigenic drift may thus result in novel viral escape strains. One of these cluster variants, disclosed as “cluster five,” has been discovered in Denmark and bears a tyrosine to phenylalanine substitution (Y453F) in the receptor-binding domain (RBD) of the spike protein (15Lassauniére R. Fonager J. Rasmussen M. Frische A. Strandh C.P. Fomsgaard V.A. Working Paper on SARS-CoV-2 Spike Mutations Arising in Danish Mink, Their 1 Spread to Humans and Neutralization Data, Preliminary Report. SSI, Copenhagen, Denmark2020: 1-7Google Scholar). This position is conserved between SARS-CoV-1 and SARS-CoV-2, and analyses of the crystal structure of SARS-CoV-2 RBD in complex with ACE-2 reveal that it is directly involved in the interaction of the virus with the host cell receptor (16Yan R. Zhang Y. Li Y. Xia L. Guo Y. Zhou Q. Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2.Science. 2021; 367: 1444-1448Crossref Scopus (3286) Google Scholar, 17Li F. Li W. Farzan M. Harrison S.C. Structure of SARS coronavirus spike receptor-binding domain complexed with receptor.Science. 2021; 309: 1864-1868Crossref Scopus (1442) Google Scholar). However, the role of this variant concerning functional transmission and establishment of immunity is unknown. At present, two new RBD variants known as N439K and N501Y have been found in humans. The latter appears to have a better transmission or improved immune evasion than the original counterpart, and this variant is currently increasing in the infection cases in the United Kingdom (18Davies N.G. Barnard R.C. Jarvis C.I. Kucharski A.J. Munday J. Pearson C.A.B. Russell T.W. Tully D.C. Abbott S. Gimma A. Waites W. Wong K.L.M. van Zandvoort K. Eggo R.M. Funk S. et al.Estimated transmissibility and impact of SARS-CoV-2 lineage B.1.1.7 in England.medRxiv. 2021; : 1-80https://doi.org/10.1101/2020.12.24.20248822Crossref Scopus (0) Google Scholar). There is a general lack of evidence characterizing the pathogenesis, functional properties, and impact on these new cluster variants' immune recognition and memory responses. Despite this, there have been speculations and reports suggesting that the cluster-five variant might have evasion potential by reducing the immunity of convalescent individuals and that it could even challenge the current global vaccine strategies (15Lassauniére R. Fonager J. Rasmussen M. Frische A. Strandh C.P. Fomsgaard V.A. Working Paper on SARS-CoV-2 Spike Mutations Arising in Danish Mink, Their 1 Spread to Humans and Neutralization Data, Preliminary Report. SSI, Copenhagen, Denmark2020: 1-7Google Scholar). Based on these presumptions, the Danish government in November 2020 decided to completely shut down all Danish mink farms, which at that time represented the largest mink fur production in the world. To address the biophysical characteristics and the impact of this variant on established immunity, we expressed recombinant SARS-CoV-2 RBD WT (WT, from the Wuhan-Hu-1/2019 isolate) and “cluster-five” Y453F and the ACE-2 ectodomain. The Y453F variant did not alter the inhibition potency of sera from convalescent individuals exposed to the WT strain in the spring of 2020, nor did it challenge the humoral vaccine response in a mouse model immunized with WT RBD or prefusion-stabilized spike protein ectodomain. However, we found a 4-fold affinity increase of the Y453F variant to the ACE-2 binding that could result in an enhanced spreading potential. To determine the biological significance of the Y453F mutation, we studied the impact on protein stability and function by thermal denaturation and binding kinetics experiments (Fig. 1). Using the ratio of the intrinsic fluorescence at 350 and 330 nm over a temperature gradient from 35 to 95 °C, we observed no significant differences in the inflection temperatures (53.43 and 53.37 °C for the WT RBD and Y453F, respectively), suggesting that the variant has no critical on protein stability (Fig. also the kinetics of the interaction with the of human ACE-2 by biolayer interferometry (Fig. and The Y453F variant with a 4-fold higher affinity than the WT and analyses of the and that it to ACE-2 and for To the tyrosine to phenylalanine substitution in the of the RBD not also recognition by the immune we the inhibition potency of sera from COVID-19 convalescent individuals a previously antibody inhibition R. M. A. L. C. L. C. M. S. T. J. P. that with SARS-CoV-2 receptor-binding domain a better neutralizing antibody response than full-length spike 2021; : Google Scholar). observed no significant between the inhibition of the two variants (Fig. and a with a significant of the antibody inhibition capacity to RBD variants (Fig. we the inhibition potency of sera and mouse from immunized with WT RBD or prefusion-stabilized spike protein as a vaccine model R. M. A. L. C. L. C. M. S. T. J. P. that with SARS-CoV-2 receptor-binding domain a better neutralizing antibody response than full-length spike 2021; : Google (Fig. from RBD immunized the WT and Y453F RBD binding to ACE-2 with an of and than sera from spike immunized of and (Fig. and of the the mouse sera did not show differences in the inhibition potency of the two RBD we the inhibition of from immunized (Fig. and analyses of the WT and Y453F RBD that the inhibition variants strongly The inhibition capacity of the The of mutations in the SARS-CoV-2 spike gene has been reported since the of The main of these mutations has been the with on the and D. G. M. the of SARS-CoV-2 and identification of mutations with the of infection and Med. 2021; PubMed Scopus Google Scholar). The of these residue are in the spike of the RBD with the mutation a variant reported on all and which to be over of L. H. A. H. A. A. M.S. Li W. T. C. Farzan M. H. SARS-CoV-2 mutation spike and Commun. 2020; 11: PubMed Scopus Google Scholar, L. Wang X. C. Wang Y. A. A. C. K. S.F. et and functional analysis of the SARS-CoV-2 spike protein 2020; Full Text Full Text PDF PubMed Scopus Google Scholar, B. S. H. J. W. N. T. B. et in SARS-CoV-2 that infectivity of the COVID-19 2020; Full Text Full Text PDF PubMed Scopus Google Scholar, J.A. Liu Y. Liu J. Xia H. Zhang X. J. D. D. Z. Z. et mutation SARS-CoV-2 2021; PubMed Scopus Google Scholar, Q. Wu J. J. Zhang L. H. Liu S. Zhao C. Zhang Q. Liu H. L. H. Wang M. Q. Li X. Sun Q. et impact of mutations in SARS-CoV-2 spike on viral infectivity and 2020; Full Text Full Text PDF PubMed Scopus Google Scholar). new genetic mutations residue in the RBD have been reported in that Y453F, and The N439K may the virus of an affinity ACE-2 a to neutralizing Q. Wu J. J. Zhang L. H. Liu S. Zhao C. Zhang Q. Liu H. L. H. Wang M. Q. Li X. Sun Q. et impact of mutations in SARS-CoV-2 spike on viral infectivity and 2020; Full Text Full Text PDF PubMed Scopus Google Scholar, W. C. Wang P. Luo M. Z. R. X. Guo Y. G. L. H. Jiang Q. N439K variant in spike protein may alter the infection and antigenicity of SARS-CoV-2 based on 2020; : Scopus (0) Google Scholar, R. A. J.A. C. L. J. S. N. R. M. N. et SARS-CoV-2 spike variant N439K fitness evading 2020; : Scopus (0) Google Scholar). The N501Y is a of a novel strain, of that has of in the spike in some of may for of the new cases at the time of (18Davies N.G. Barnard R.C. Jarvis C.I. Kucharski A.J. Munday J. Pearson C.A.B. Russell T.W. Tully D.C. Abbott S. Gimma A. Waites W. Wong K.L.M. van Zandvoort K. Eggo R.M. Funk S. et al.Estimated transmissibility and impact of SARS-CoV-2 lineage B.1.1.7 in England.medRxiv. 2021; : 1-80https://doi.org/10.1101/2020.12.24.20248822Crossref Scopus (0) Google Scholar). The Y453F in Denmark in the of 2020 among farmed minks. The variant to the “cluster mutation (15Lassauniére R. Fonager J. Rasmussen M. Frische A. Strandh C.P. Fomsgaard V.A. Working Paper on SARS-CoV-2 Spike Mutations Arising in Danish Mink, Their 1 Spread to Humans and Neutralization Data, Preliminary Report. SSI, Copenhagen, Denmark2020: 1-7Google Scholar) and arose a it reported to have been to humans. of minks and down a However, a functional characterization of the impact of the Y453F RBD variant on immunity and ACE-2 interaction has not been we the interaction with COVID-19 convalescent sera from individuals that have been infected with the original SARS-CoV-2 variant, we found no in the capacity to the binding of the Y453F variant to This with a that found that from convalescent individuals of a of with and a potency the variant (15Lassauniére R. Fonager J. Rasmussen M. Frische A. Strandh C.P. Fomsgaard V.A. Working Paper on SARS-CoV-2 Spike Mutations Arising in Danish Mink, Their 1 Spread to Humans and Neutralization Data, Preliminary Report. SSI, Copenhagen, Denmark2020: 1-7Google Scholar). The major in the the of is that the from et al. (15Lassauniére R. Fonager J. Rasmussen M. Frische A. Strandh C.P. Fomsgaard V.A. Working Paper on SARS-CoV-2 Spike Mutations Arising in Danish Mink, Their 1 Spread to Humans and Neutralization Data, Preliminary Report. SSI, Copenhagen, Denmark2020: 1-7Google Scholar) the we have the inhibition of the interaction To the possible inhibition differences between the two RBD variants, we a vaccine immunized with WT RBD or the WT spike and the antibody and capacity of WT and Y453F the vaccine we found no concerning the inhibition of the two variants, which also the we neutralizing raised WT RBD or spike antigens. The tyrosine at position has been shown to be a critical residue in interaction with the ACE-2 receptor the (16Yan R. Zhang Y. Li Y. Xia L. Guo Y. Zhou Q. Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2.Science. 2021; 367: 1444-1448Crossref Scopus (3286) Google Scholar). that there would be a or affinity with the substitution to a However, to we found a higher affinity of the RBD binding to ACE-2 with WT The for this to be there are other tyrosine in RBD that might be in the ACE-2 The of this affinity increase on the could even be for the However, since findings are be the transmission capacity of this SARS-CoV-2 results two regarding the developing of spike that immunity might not be and that also be on characterizing the transmission capacity and interaction properties of new SARS-CoV-2 strains. This in the of the new discovered N501Y that a time transmission in many including by represented than of the cases K. coronavirus in the United Kingdom 2020; Google Scholar). The functional properties of the N501Y variant are not established. these results that the of the SARS-CoV-2 virus has been it in the of receptor affinity adaptation and rather than an immune evasion strategy. The to the SARS-CoV-2 Y453F RBD variant with a and a by Y453F RBD by to the On the and the protein by 2 at temperature with of of by and to the The protein to a in RBD Y453F and on the with a The to the of human ACE-2 with a and a human by and a by to the On 5 the and the protein on a by on a in The and of the human ACE-2 receptor and SARS-CoV-2 WT RBD have been R. M. A. L. C. L. C. M. S. T. J. P. that with SARS-CoV-2 receptor-binding domain a better neutralizing antibody response than full-length spike 2021; : Google Scholar). The impact of the Y453F substitution on protein stability on a RBD WT and Y453F in to a thermal of in denaturation from the intrinsic fluorescence at 350 and 330 temperatures a by the from the kinetics experiments on an in the and with (1Shi J. Wen Z. Zhong G. Yang H. Wang C. Huang B. Liu R. He X. Shuai L. Sun Z. Zhao Y. Liu P. Liang L. Cui P. Wang J. et al.Susceptibility of ferrets, cats, dogs, and other domesticated animals to SARS-coronavirus 2.Science. 2020; 368: 1016-1020Crossref PubMed Scopus (1182) Google Scholar), on P.J. Hatta M. Chiba S. Maemura T. Fan S. Takeda M. Kinoshita N. Hattori S. Sakai-Tagawa Y. Iwatsuki-Horimoto K. Imai M. Kawaoka Y. Transmission of SARS-CoV-2 in domestic cats.N. Engl. J. Med. 2020; 383: 592-594Crossref PubMed Scopus (331) Google Scholar), S.F. Yan L.-M. Chin A.W.H. Fung K. Choy K.-T. Wong A.Y.L. Kaewpreedee P. Perera R.A.P.M. Poon L.L.M. Nicholls J.M. Peiris M. Yen H.-L. Pathogenesis and transmission of SARS-CoV-2 in golden hamsters.Nature. 2020; 583: 834-838Crossref PubMed Scopus (827) Google Scholar), to at for WT RBD or for Y453F RBD and (4Oreshkova N. Molenaar R.J. Vreman S. Harders F. Oude Munnink B.B. Hakze-van der Honing R.W. Gerhards N. Tolsma P. Bouwstra R. Sikkema R.S. Tacken M.G. de Rooij M.M. Weesendorp E. Engelsma M.Y. Bruschke C.J. et al.SARS-CoV-2 infection in farmed minks, The Netherlands, April and May 2020.Euro Surveill. 2020; 25: 2001005Crossref PubMed Scopus (426) Google Scholar) the for with and the and The from The and and to a binding The inhibition potency of COVID-19 convalescent immunized mouse and an in antibody inhibition ELISA-based R. M. A. L. C. L. C. M. S. T. J. P. that with SARS-CoV-2 receptor-binding domain a better neutralizing antibody response than full-length spike 2021; : Google Scholar). with 1 of ACE-2 at °C in The for 1 in with a of WT or Y453F RBD with in with as COVID-19 convalescent sera in a immunized mouse sera in an 4-fold at and in a 4-fold at the to ACE-2 and for The with One for and with The read at the with all in an at of from convalescent with a previously SARS-CoV-2 infection in the study. The have been L. L. M. A. C. T. C. K. Bayarri-Olmos R. P. SARS-CoV-2 antibody are to disease in COVID-19 convalescent Immunol. 2021; PubMed Scopus Google Scholar). from individuals as the control. The of convalescent in this has been by the of the of Denmark with the The human to the of The animal in this have been by the Danish with the analyses with The response with the to the and to 1 and The between the inhibition potency of sera and the WT and Y453F RBD by the analyses as and as and a significance of for WT and the Y453F RBD with the data are this The that have no of interest with the of this The would like to and from for analysis of the recombinant and and from the of for R. and S. and the R. and C. H. recombinant protein R. A. L. B. and S. R. L. B. and S. the R. P. and S. the with from all the of the This by from the to R. and P. and the and to R. and P.