In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...

Institut Pasteur

The rabies virus glycoprotein molecule (G) can be divided into two parts separated by a flexible hinge: the NH2 half (site II part) containing antigenic site II up to the linear region (amino acids [aa] 253 to 275 encompassing epitope VI [aa 264]) and the COOH half (site III part) containing antigenic site III and the transmembrane and cytoplasmic domains. The structural and immunological roles of each part were investigated by cell transfection and mouse DNA-based immunization with homogeneous and chimeric G genes formed by fusion of the site II part of one genotype (GT) with the site III part of the same or another GT. Various site II-site III combinations between G genes of PV (Pasteur virus strain) rabies (GT1), Mokola (GT3), and EBL1 (European bat lyssavirus 1 [GT5]) viruses were tested. Plasmids pGPV-PV, pGMok-Mok, pGMok-PV, and pGEBL1-PV induced transient expression of correctly transported and folded antigens in neuroblastoma cells and virus-neutralizing antibodies against parental viruses in mice, whereas, pG-PVIII (site III part only) and pGPV-Mok did not. The site III part of PV (GT1) was a strong inducer of T helper cells and was very effective at presenting the site II part of various GTs. Both parts are required for correct folding and transport of chimeric G proteins which have a strong potential value for immunological studies and development of multivalent vaccines. Chimeric plasmid pGEBL1-PV broadens the spectrum of protection against European lyssavirus genotypes (GT1, GT5, and GT6).

Chimeric lyssavirus glycoproteins with increased immunological potential.

The low-affinity nerve-growth factor receptor p75NTR interacts in vitro with the rabies virus (RV) glycoprotein and serves as a receptor for RV. The Lyssavirus genus comprises seven genotypes (GTs) of rabies and rabies-related viruses. The ability of p75NTR to interact with the glycoprotein of representative lyssaviruses from each GT was investigated. This investigation was based on a specific binding assay between BSR cells infected with a lyssavirus and Spodoptera frugiperda (Sf21) cells expressing p75NTR on the cell surface. A specific interaction was observed with the glycoprotein of GT 1 RV (challenge virus standard or Pasteur virus strains) as well as wild-type RV and the glycoprotein of GT 6 European bat lyssavirus type 2. In contrast, no interaction was detected with the glycoprotein of lyssaviruses of GTs 2-5 and 7. Therefore, p75NTR is only a receptor for some lyssavirus glycoproteins, indicating that the other GTs must use an alternative specific receptor.

Interaction of lyssaviruses with the low-affinity nerve-growth factor receptor p75NTR.

We have developed a new strategy for antiviral peptide discovery by using lyssaviruses (rabies virus and rabies-related viruses) as models. Based on the mimicry of natural bioactive peptides, two genetically encoded combinatorial peptide libraries composed of intrinsically constrained peptides (coactamers) were designed. Proteomic knowledge concerning the functional network of interactions in the lyssavirus transcription-replication complex highlights the phosphoprotein (P) as a prime target for inhibitors of viral replication. We present an integrated, sequential drug discovery process for selection of peptides with antiviral activity directed against the P. Our approach combines (i). an exhaustive two-hybrid selection of peptides binding two phylogenetically divergent lyssavirus P's, (ii). a functional analysis of protein interaction inhibition in a viral reverse genetic assay, coupled with a physical analysis of viral nucleoprotein-P complex by protein chip mass spectrometry, and (iii). an assay for inhibition of lyssavirus infection in mammalian cells. The validity of this strategy was demonstrated by the identification of four peptides exhibiting an efficient antiviral activity. Our work highlights the importance of P as a target in anti-rabies virus drug discovery. Furthermore, the screening strategy and the coactamer libraries presented in this report could be considered, respectively, a general target validation strategy and a potential source of biologically active peptides which could also help to design pharmacologically active peptide-mimicking molecules. The strategy described here is easily applicable to other pathogens.

Antiviral drug discovery strategy using combinatorial libraries of structurally constrained peptides.

Nous avons d&eacute;velopp&eacute; une nouvelle strat&eacute;gie pour la d&eacute;couverte de peptide antiviral en utilisant des Lyssavirus (virus de la rage et li&eacute;es &agrave; la rage virus) comme mod&egrave;les. Bas&eacute; sur le mim&eacute;tisme des peptides bioactifs naturels, deux cod&eacute; g&eacute;n&eacute;tiquement combinatoire peptide biblioth&egrave;ques compos&eacute;s de peptides intrins&egrave;quement limit&eacute;es (coactamers) ont &eacute;t&eacute; con&ccedil;us. Connaissances de prot&eacute;omique concernant le r&eacute;seau fonctionnel des interactions dans le complexe de transcription-r&eacute;plication de lyssavirus met en &eacute;vidence la phosphoprot&eacute;ine (P) comme une cible de choix pour les inhibiteurs de la r&eacute;plication virale. Nous pr&eacute;sentons un processus de d&eacute;couverte de drogue s&eacute;quentielles, int&eacute;gr&eacute;e pour la s&eacute;lection des peptides avec une activit&eacute; antivirale dirig&eacute;e contre le P. Notre approche combine (i). une s&eacute;lection exhaustive de deux-hybride de peptides liant deux lyssavirus phylog&eacute;n&eacute;tiquement divergentes P, (ii). une analyse fonctionnelle de l&#39;inhibition de la prot&eacute;ine interaction dans un test g&eacute;n&eacute;tique inverse virale, de pair avec une analyse physique de nucl&eacute;oprot&eacute;ine virale-P complexe prot&eacute;ine puce spectrom&eacute;trie de masse et (iii). un test d&#39;inhibition de l&#39;infection de lyssavirus en cellules mammif&egrave;res. La validit&eacute; de cette strat&eacute;gie a &eacute;t&eacute; d&eacute;montr&eacute;e par l&#39;identification de quatre peptides pr&eacute;sentant une activit&eacute; antivirale efficace. Notre travail souligne l&#39;importance de P comme cible dans la d&eacute;couverte de m&eacute;dicaments virus antirabique. En outre, la strat&eacute;gie de d&eacute;pistage et les biblioth&egrave;ques de coactamer pr&eacute;sent&eacute;s dans ce rapport sont envisageables, respectivement, une strat&eacute;gie de validation de cibles g&eacute;n&eacute;rales et une source potentielle de peptides biologiquement actifs qui pourraient &eacute;galement contribuer &agrave; concevoir des mol&eacute;cules pharmacologiquement actives imitant peptide. La strat&eacute;gie d&eacute;crite ici est facilement applicable aux autres agents pathog&egrave;nes.

Strat&eacute;gie de d&eacute;couverte de m&eacute;dicaments antiviraux &agrave; l&#39;aide de biblioth&egrave;ques combinatoires de peptides structurellement contraints.

Abbiamo sviluppato una nuova strategia per la scoperta del peptide antivirali utilizzando lyssaviruses (virus della rabbia e virus correlati a rabbia) come modelli. Basato sul mimetismo di peptidi bioattivi naturali, due geneticamente codificati combinatorial peptide librerie composte di peptidi intrinsecamente vincolati (coactamers) sono state progettate. Proteomic conoscenze riguardanti la rete funzionale delle interazioni del complesso di trascrizione-replica lyssavirus mette in evidenza la fosfoproteina (P) come una destinazione privilegiata per gli inibitori della replicazione virale. Vi presentiamo un processo di scoperta di droga integrato, sequenziale per la selezione dei peptidi con attivit&agrave; antivirale diretto contro la P. Il nostro approccio unisce (i). un&#39;esaustiva selezione due-ibrido di peptidi associazione lyssavirus filogeneticamente divergenti due P, (ii). un&#39;analisi funzionale di inibizione di interazione della proteina in un&#39;analisi genetica inversa virale, accoppiato con un&#39;analisi fisica della nucleoproteina virale-P complesso di spettrometria di massa di chip della proteina e (iii). un test per l&#39;inibizione di lyssavirus infezione in cellule di mammifero. La validit&agrave; di questa strategia &egrave; stata dimostrata dall&#39;identificazione delle quattro peptidi esibendo un&#39;efficiente attivit&agrave; antivirale. Il nostro lavoro evidenzia l&#39;importanza di P come bersaglio nella scoperta della droga virus antirabbico. Inoltre, la strategia di screening e le librerie di coactamer presentate in questo rapporto potrebbero essere considerate, rispettivamente, una strategia di convalida target generale e una potenziale fonte di peptidi biologicamente attivi che potrebbe anche contribuire a progettare molecole farmacologicamente attive del peptide-imitando. La strategia qui descritta &egrave; facilmente applicabile agli altri agenti patogeni.

Strategia di scoperta del farmaco antivirale utilizzando librerie combinatorie dei peptidi strutturalmente vincolate.

We wanted to develop a therapeutic approach against rabies disease by targeting the lyssavirus transcription/replication complex. Because this complex (nucleoprotein N-RNA template processed by the L polymerase and its cofactor, the phosphoprotein P) is similar to that of other negative-strand RNA viruses, we aimed to design broad-spectrum antiviral drugs that could be used as a complement to postexposure vaccination and immunotherapy. Recent progress in understanding the structure/function of the rabies virus P, N, and L proteins predicts that the amino-terminal end of P is an excellent target for destabilizing the replication complex because it interacts with both L (for positioning onto the N-RNA template) and N (for keeping N soluble, as needed for viral RNA encapsidation). Thus, peptides mimicking various lengths of the amino-terminal end of P have been evaluated, as follows: (i) for binding properties to the N-P-L partners by the two-hybrid method; (ii) for their capacity to inhibit the transcription/replication of a rabies virus minigenome encoding luciferase in BHK-21-T7 cells; and (iii) for their capacity to inhibit rabies virus infection of BHK-21-T7 cells and of two derivatives of the neuronal SK-N-SH cell line. Peptides P60 and P57 (the first 60 and first 57 NH2 residues of P, respectively) exhibited a rapid, strong, and long-lasting inhibitory potential on luciferase expression (>95% from 24 h to 55 h). P42 was less efficient in its inhibition level (75% for 18 to 30 h) and duration (40% after 48 h). The most promising peptides were synthesized in tandem with the Tat sequence, allowing cell penetration. Their inhibitory effects were observed on BHK-21-T7 cells infected with rabies virus and Lagos bat virus but not with vesicular stomatitis virus. In neuronal cells, a significant inhibition of both nucleocapsid inclusions and rabies virus release was observed.

Peptides that mimic the amino-terminal end of the rabies virus phosphoprotein have antiviral activity.

Nous avons voulu d&eacute;velopper une approche th&eacute;rapeutique contre la maladie de la rage en ciblant la transcription/r&eacute;plication de lyssavirus complexe. Parce que ce complexe (mod&egrave;le de nucl&eacute;oprot&eacute;ine N-RNA trait&eacute;e par la polym&eacute;rase L et son cofacteur, la phosphoprot&eacute;ine P) est similaire &agrave; celle des autres virus &agrave; ARN n&eacute;gatif-brin, nous avons cherch&eacute; &agrave; concevoir des m&eacute;dicaments antiviraux &agrave; large spectre qui pourraient &ecirc;tre utilis&eacute;s comme un compl&eacute;ment &agrave; la vaccination post-exposition et immunoth&eacute;rapie. Les progr&egrave;s r&eacute;cents dans la compr&eacute;hension de la structure/fonction des prot&eacute;ines P, N et L la rage virus pr&eacute;dit que l&#39;extr&eacute;mit&eacute; amino-terminale de P est une excellente cible pour d&eacute;stabiliser la r&eacute;plication complexe car elle interagit avec L (pour le positionnement sur le mod&egrave;le N-RNA) et N (pour maintien N soluble, selon les besoins pour l&#39;encapsidation de RNA virale). Ainsi, les peptides mimant les diff&eacute;rentes longueurs de l&#39;extr&eacute;mit&eacute; amino-terminale de P ont &eacute;t&eacute; &eacute;valu&eacute;es, comme suit: (i) pour lier des propri&eacute;t&eacute;s &agrave; des partenaires de N-P-L par la m&eacute;thode de double-hybride ; (ii) pour leur capacit&eacute; &agrave; inhiber la transcription/r&eacute;plication d&#39;un minigenome de virus de la rage codant la lucif&eacute;rase dans les cellules BHK-21-T7 ; et (iii) pour leur capacit&eacute; &agrave; inhiber l&#39;infection de virus de la rage de cellules BHK-21-T7 et de deux d&eacute;riv&eacute;s de la neuronale SK-N-SH lign&eacute;e cellulaire. Peptides P60 et P57 (les premiers 60 premiers 57 NH2 r&eacute;sidus et de P, respectivement) pr&eacute;sentait un potentiel inhibiteur rapid, fort et durable sur l&#39;expression de la lucif&eacute;rase (&gt; 95 % de 24h &agrave; 55 h). P42 &eacute;tait moins efficace dans son niveau d&#39;inhibition (75 % pour les 18 &agrave; 30 h) et la dur&eacute;e (40 % apr&egrave;s 48 h). Les peptides plus prometteurs ont &eacute;t&eacute; synth&eacute;tis&eacute;s en tandem avec la s&eacute;quence de la Tat, qui permet la p&eacute;n&eacute;tration de la cellule. Leurs effets inhibiteurs ont &eacute;t&eacute; observ&eacute;s sur les cellules BHK-21-T7 infect&eacute;es avec le virus de la rage et le virus de chauve-souris de Lagos, mais pas par le virus de la stomatite v&eacute;siculeuse. Dans les cellules neuronales, une inhibition significative des inclusions de nucl&eacute;ocapside et lib&eacute;ration de virus de la rage a &eacute;t&eacute; observ&eacute;e.

Les peptides qui imitent l&#39;extr&eacute;mit&eacute; amino-terminale de la phosphoprot&eacute;ine de virus de la rage ont une activit&eacute; antivirale.

Abbiamo voluto sviluppare un approccio terapeutico contro la malattia della rabbia di mira la trascrizione/replica di lyssavirus complesso. Perch&eacute; questo complesso (modello nucleoproteina N-RNA elaborato dalla polimerasi L e suo cofattore, la fosfoproteina P) &egrave; simile a quella di altri virus a RNA negativo, abbiamo mirato a progettare farmaci antivirali ad ampio spettro che potrebbero essere utilizzati come un complemento alla vaccinazione postexposure e immunoterapia. Recenti progressi nella comprensione della struttura/funzione delle proteine P, N e L del virus di rabbia predice che la fine del amminico-terminale di P &egrave; un&#39;eccellente destinazione per destabilizzare la replica complesso perch&eacute; interagisce con L (per il posizionamento sul modello N-RNA) e N (per mantenere N solubile, come necessario per encapsidation di RNA virale). Cos&igrave;, peptidi imitando varie lunghezze dell&#39;estremit&agrave; amino-terminale del P sono state valutate, come segue: (i) per l&#39;associazione di propriet&agrave; per i partner di N-P-L con il metodo del due-ibrido; (ii) per la loro capacit&agrave; di inibire la trascrizione/replica di un minigenome del virus di rabbia codifica luciferase nelle cellule BHK-21-T7; e (iii) per la loro capacit&agrave; di inibire l&#39;infezione del virus di rabbia delle cellule BHK-21-T7 e di due derivati di un neurone SK-N-SH cella linea. Peptidi P60 e P57 (i primi primi e 60 57 NH2 residui di P, rispettivamente) esposto un potenziale rapido, forte e duraturo inibitorio sull&#39;espressione luciferase (&amp;gt; 95% da 24 h a h 55). P42 era meno efficiente nel suo livello di inibizione (75% per 18 a 30 h) e la durata (40% dopo 48 h). I peptidi pi&ugrave; promettenti sono stati sintetizzati in tandem con la sequenza di Tat, permettendo la penetrazione delle cellule. Loro effetti inibitori sono stati osservati su cellule BHK-21-T7 infettate con il virus della rabbia e Lagos bat virus ma non con virus di stomatite vescicolare. Nelle cellule neuronali, &egrave; stata osservata una significativa inibizione di rilascio di virus di rabbia e di inclusioni nucleocapside.

Peptidi che mimano la fine del amminico-terminale della fosfoproteina di virus della rabbia hanno attivit&agrave; antivirale.

Hantaviruses are emerging zoonotic viruses that cause human diseases. In this study, sera from 642 mammals from La Réunion and Mayotte islands (Indian Ocean) were screened for the presence of hantaviruses by molecular analysis. None of the mammals from La Réunion island was positive, but hantavirus genomic RNA was discovered in 29/160 (18 %) Rattus rattus from Mayotte island. The nucleoprotein coding region was sequenced from the liver and spleen of all positive individuals allowing epidemiological and intra-strain variability analyses. Phylogenetic analysis based on complete coding genomic sequences showed that this Murinae-associated hantavirus is a new variant of Thailand virus. Further studies are needed to investigate hantaviruses in rodent hosts and in Haemorrhagic Fever with Renal Syndrome (HFRS) human cases.

Discovery of hantavirus circulating among Rattus rattus in French Mayotte island, Indian Ocean.

Over the last 20 years, natural peptides playing a key role in defense mechanisms and innate immunity have been isolated from unicellular organisms. Amphibian skin secretes dermaseptins, 24-34 amino acids in length that have a wide antimicrobial spectrum incorporating yeast, fungi, protozoa, bacteria and enveloped viruses. The anti-rabies virus (RABV) activity of dermaseptins S3 (30aa) and S4 (28aa) from Phyllomedusa sauvagei has been investigated, and further dissected its molecular basis by comparing punctual mutation or deletion of S4 analogues. The results showed that: (1) S4 is more active than S3 against RABV infection, 89% versus 38% inhibition at 7.5 μM; (2) the 5 NH2-aa of S4 are crucial for its inhibitory potential (S4 lost any inhibition) but the COOH terminus stabilizes the inhibitory potential (S4 showed only 23% inhibition at 7.5 μM); (3) there is a correlation between viral inhibition and dermaseptin cytotoxicity, which remains however moderated for BSR cells (≤12% at 10 μM). A single mutation in position 4 (S4) slightly reduced cytotoxicity while keeping its antiviral activity, 97% at 7.5 μM. S4 and S4 showed an antiviral activity in vitro when provided 1 h after infection. In vivo experiments in mice by intramuscular injection of non-toxic doses of dermaseptin S4 1 h post-infection by a lethal dose of RABV at the same site allowed more than 50% improvement in mice survival. This study highlights the potential interest of dermaseptins as non-expansive alternatives to rabies immunoglobulins for the treatment of rabies that continues to claim about 60,000 human lives per year worldwide, almost exclusively in developing countries.

Corinne Jallet

Unit Antiviral Strategies,
OIE Reference Laboratories for Rift Valley Fever Virus & Crimean Congo Hemorrhagic Fever Virus,
Unit Antiviral Strategies, OIE Reference Laboratories for Rift Valley Fever Virus & Crimean Congo Hemorrhagic Fever Virus

Institut Pasteur

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency