US20190070284A1

US20190070284A1 - Pan South American Arenavirus Live Attenuated Vaccine

Info

Publication number: US20190070284A1
Application number: US16/083,501
Authority: US
Inventors: Joseph W. Golden; Jay W. Hooper
Original assignee: US Department of Army
Current assignee: US Department of Army
Priority date: 2015-08-28
Filing date: 2017-08-30
Publication date: 2019-03-07
Also published as: US9870761B2; US20170061942A1

Abstract

Disclosed herein are methods of making attenuated Arenaviruses, compositions comprising the attenuated Arenaviruses, and methods of using the attenuated Arenaviruses. As disclosed herein, the attenuated Arenaviruses have a deletion of at least a portion of the intergenic region (IGR) of one or both genome segments.

Description

ACKNOWLEDGEMENT OF GOVERNMENT SUPPORT

This invention was made by employees of the United States Army Medical Research and Materiel Command, which is an agency of the United States Government. The Government has certain rights in the invention.

REFERENCE TO A SEQUENCE LISTING SUBMITTED VIA EFS-WEB

The content of the ASCII text file of the sequence listing named “20170818_034047_067WO1_seq_ST25” which is 1.25 kb in size was created on Aug. 18, 2017, and electronically submitted via EFS-Web herewith the application is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention generally relates to Arenaviruses and vaccines.

2. Description of the Related Art

Members of the Arenaviridae (Arenaviruses) are enveloped ambisense single-stranded RNA viruses with two segments, small (S) and large (L), encoding a 10.7 kb genome expressing five distinct proteins. The L-segment encodes the matrix ring finger Z protein4 and the polymerase L protein. The S-segment encodes the nucleoprotein (NP) and the glycoprotein precursor (GPC). GPC is cleaved into two glycoproteins, GP1 and GP2 by the cellular protease S1P. Each RNA segment encodes two ORFs and contains noncoding regions including 5′ and 3′ untranslated regions (UTRs) and a non-coding intergenic region (IGR). Mammalian Arenaviruses (genus mammarenavirus) are divided into the Old World Complex Arenaviruses (OW Arenaviruses) and New World Complex Arenaviruses (NW Arenaviruses) based on geographical distribution and serology.
Machupo virus (MACV) is a member of the NW Arenaviruses and is the causative agent of Bolivian hemorrhagic fever [1]. Human infections result from exposure to chronically infected rodents (Calomys callosus) [2], but human-to-human spread has been reported [3]. Bolivian hemorrhagic fever is a febrile illness often associated with vascular leakage and occasional concomitant neurological manifestations [3, 4]. Infection can result in a systemic inflammatory response syndrome leading to multiple organ failure and death. Within the NW Arenaviruses are several other Arenaviruses that cause hemorrhagic fever (HF) in humans include the Junin virus (JUNV) the causative agent of Argentine hemorrhagic fever and the Guanarito virus (GTOV) the causative agent of Venezuelan hemorrhagic fever [5]. Other pathogenic Arenaviruses that cause HF in humans, including the Sabia virus, the Chapare virus, and the Whitewater arroyo virus, have more recently emerged [6-8]. Accordingly, Arenaviruses are an important group of emerging and re-emerging human pathogens.
Active and passive vaccine strategies, as well as small molecule inhibitors have been shown to be effective at reducing lethality in humans [9-11]. An attenuated live-virus vaccine derived from JUNV, termed Candid#1, is currently used in populations at high risk to JUNV infection [12-15]. Implementation of Candid#1 in the endemic region of Argentina reduced fatality rates substantially. Some evidence based on studies in guinea pigs and non-human primates (NHPs) suggest that Candid#1 can cross-protect against MACV [16], however these findings have not been validated in humans. Candid#1 was produced by passage of the virulent strain XJ twice in guinea pigs, 44 times in mouse brains, and finally several passages in fetal rhesus lung diploid cells (FRhL-2) [15, 17-19]. This process produced a strain that is attenuated in humans, non-human primates, guinea pigs, and mice and also lacks neurotropism in animal models. The exact nature of the attenuation is enigmatic and recent evidence indicates that single amino acid changes in the glycoprotein 2 transmembrane region restores virulence in neonatal mice [17]. Other NW Arenavirus vaccine strategies have included the use of Tacaribe virus (TACV), an Arenavirus serologically related to JUNV and MACV believed to be apathogenic in humans. In animal models, TACV functions as a vaccine against JUNV [20, 21]. However, the underlying mechanism(s) attenuating TACV in humans is unclear. Glycoprotein-targeting subunit vaccines based on modified vaccine Ankara or Venezuelan equine encephalitis replicon vectored systems protect against lethal infection by JUNV in guinea pig models [22, 23]. However, because of heterogeneity in the glycoproteins [20, 24, 25], it is unlikely these vaccines will provide sufficient cross-protection against heterologous Arenaviruses. Thus, alternative strategies aimed at producing safe and broadly protective Arenavirus vaccines are needed.

SUMMARY OF THE INVENTION

In some embodiments, the present invention provides a method of making an IGR Deletion Arenavirus, which comprises deleting at least a portion of an intergenic region (IGR) of one or both genome segments of an Arenavirus. In some embodiments, the portion of the IGR is deleted using recombinant DNA methods known in the art. In some embodiments, the genome segment is the L-segment. In some embodiments, the genome segment is the S-segment. In some embodiments, at least about 15-50 nucleotide bases of the IGR are deleted. In some embodiments, at least about 20-45 nucleotide bases of the IGR are deleted. In some embodiments, at least about 25-40 nucleotide bases of the IGR are deleted. In some embodiments, at least about 30-35 nucleotide bases of the IGR are deleted. In some embodiments, at least about 20%, preferably at least about 30%, more preferably at least about 40%, even more preferably at least about 50%, and most preferably about 60% of the nucleotide bases of the IGR are deleted. In some embodiments, the native structure of the hairpin loop formed by the nucleotide sequence of the IGR of the Arenavirus is altered by the deletion of the nucleotide bases. In some embodiments, the Arenavirus is an OW Arenavirus. In some embodiments, the Arenavirus is a NW Arenavirus. In some embodiments, the Arenavirus is a Lassa virus, a Lujo virus, a Lymphocytic choriomeningitis virus, a Chapare virus, a Guanarito virus, a Junin virus, a Machupo virus, a Sabia virus, a Tacaribe virus, or a Whitewater Arroyo virus. In some embodiments, the Arenavirus is a Junin virus, a Machupo virus, a Sabia virus, or a Tacaribe virus. In some embodiments, the Arenavirus is JUNV strain Candid#1. In some embodiments, the Arenavirus is MACV strain Carvallo. In some embodiments, the Arenavirus is MACV strain Chicava.
In some embodiments, the present invention provides a composition comprising, consisting essentially of, or consisting of one or more IGR Deletion Arenaviruses, which have at least a portion of an intergenic region (IGR) of one or both genome segments of an Arenavirus. In some embodiments, the portion of the IGR is deleted using recombinant DNA methods known in the art. In some embodiments, the genome segment is the L-segment. In some embodiments, the genome segment is the S-segment. In some embodiments, at least about 15-50 nucleotide bases of the IGR are deleted. In some embodiments, at least about 20-45 nucleotide bases of the IGR are deleted. In some embodiments, at least about 25-40 nucleotide bases of the IGR are deleted. In some embodiments, at least about 30-35 nucleotide bases of the IGR are deleted. In some embodiments, at least about 20%, preferably at least about 30%, more preferably at least about 40%, even more preferably at least about 50%, and most preferably about 60% of the nucleotide bases of the IGR are deleted. In some embodiments, the native structure of the hairpin loop formed by the nucleotide sequence of the IGR of the Arenavirus is altered by the deletion of the nucleotide bases. In some embodiments, the Arenavirus is an OW Arenavirus. In some embodiments, the Arenavirus is a NW Arenavirus. In some embodiments, the Arenavirus is a Lassa virus, a Lujo virus, a Lymphocytic choriomeningitis virus, a Chapare virus, a Guanarito virus, a Junin virus, a Machupo virus, a Sabia virus, a Tacaribe virus, or a Whitewater Arroyo virus. In some embodiments, the Arenavirus is a Junin virus, a Machupo virus, a Sabia virus, or a Tacaribe virus. In some embodiments, the Arenavirus is JUNV strain Candid#1. In some embodiments, the Arenavirus is MACV strain Carvallo. In some embodiments, the Arenavirus is MACV strain Chicava. In some embodiments, the composition comprises a pharmaceutically acceptable carrier. In some embodiments, the composition comprises an adjuvant.
In some embodiments, the present invention provides a method of treating, inhibiting, or reducing an Arenavirus infection or a disease caused by an Arenavirus in a subject, which comprises administering to the subject an effective amount of one or more IGR Deletion Arenaviruses, which have at least a portion of an intergenic region (IGR) of one or both genome segments of an Arenavirus. In some embodiments, the portion of the IGR is deleted using recombinant DNA methods known in the art. In some embodiments, the genome segment is the L-segment. In some embodiments, the genome segment is the S-segment. In some embodiments, at least about 15-50 nucleotide bases of the IGR are deleted. In some embodiments, at least about 20-45 nucleotide bases of the IGR are deleted. In some embodiments, at least about 25-40 nucleotide bases of the IGR are deleted. In some embodiments, at least about 30-35 nucleotide bases of the IGR are deleted. In some embodiments, at least about 20%, preferably at least about 30%, more preferably at least about 40%, even more preferably at least about 50%, and most preferably about 60% of the nucleotide bases of the IGR are deleted. In some embodiments, the native structure of the hairpin loop formed by the nucleotide sequence of the IGR of the Arenavirus is altered by the deletion of the nucleotide bases. In some embodiments, the Arenavirus is an OW Arenavirus. In some embodiments, the Arenavirus is a NW Arenavirus. In some embodiments, the Arenavirus is a Lassa virus, a Lujo virus, a Lymphocytic choriomeningitis virus, a Chapare virus, a Guanarito virus, a Junin virus, a Machupo virus, a Sabia virus, a Tacaribe virus, or a Whitewater Arroyo virus. In some embodiments, the Arenavirus is a Junin virus, a Machupo virus, a Sabia virus, or a Tacaribe virus. In some embodiments, the Arenavirus is JUNV strain Candid#1. In some embodiments, the Arenavirus is MACV strain Carvallo. In some embodiments, the Arenavirus is MACV strain Chicava. In some embodiments, the Arenavirus and the IGR Deletion Arenavirus are of the same viral species. In some embodiments, the Arenavirus and the IGR Deletion Arenavirus are of different viral species. In some embodiments, the Arenavirus is an OW Arenavirus and the IGR Deletion Arenavirus is a NW Arenavirus. In some embodiments, the Arenavirus is a NW Arenavirus and the IGR Deletion Arenavirus is an OW Arenavirus. In some embodiments, the Arenavirus and the IGR Deletion Arenavirus are each independently selected from the group consisting of Lassa virus, Lujo virus, Lymphocytic choriomeningitis virus, Chapare virus, Guanarito virus, Junin virus, Machupo virus, Sabia virus, Tacaribe virus, and Whitewater Arroyo virus. In some embodiments, the Arenavirus is a Lassa virus, a Lujo virus, a Lymphocytic choriomeningitis virus, a Chapare virus, a Guanarito virus, a Junin virus, a Machupo virus, a Sabia virus, a Tacaribe virus, or a Whitewater Arroyo virus and the IGR Deletion Arenavirus is JUNV strain Candid#1, MACV strain Carvallo, or MACV strain Chicava, and has a portion of its intragenic region (IGR) of the L-segment deleted. In some embodiments, the Arenavirus is a Guanarito virus or a Junin virus and the IGR Deletion Arenavirus is a Machupo virus. In some embodiments, the one or more IGR Deletion Arenaviruses are administered in the form of a composition as described herein.
In some embodiments, the present invention provides a method of immunizing a subject against an Arenavirus, which comprises administering to the subject an immunogenic amount of one or more IGR Deletion Arenaviruses, which have at least a portion of an intergenic region (IGR) of one or both genome segments of an Arenavirus. In some embodiments, the portion of the IGR is deleted using recombinant DNA methods known in the art. In some embodiments, the genome segment is the L-segment. In some embodiments, the genome segment is the S-segment. In some embodiments, at least about 15-50 nucleotide bases of the IGR are deleted. In some embodiments, at least about 20-45 nucleotide bases of the IGR are deleted. In some embodiments, at least about 25-40 nucleotide bases of the IGR are deleted. In some embodiments, at least about 30-35 nucleotide bases of the IGR are deleted. In some embodiments, at least about 20%, preferably at least about 30%, more preferably at least about 40%, even more preferably at least about 50%, and most preferably about 60% of the nucleotide bases of the IGR are deleted. In some embodiments, the native structure of the hairpin loop formed by the nucleotide sequence of the IGR of the Arenavirus is altered by the deletion of the nucleotide bases. In some embodiments, the Arenavirus is an OW Arenavirus. In some embodiments, the Arenavirus is a NW Arenavirus. In some embodiments, the Arenavirus is a Lassa virus, a Lujo virus, a Lymphocytic choriomeningitis virus, a Chapare virus, a Guanarito virus, a Junin virus, a Machupo virus, a Sabia virus, a Tacaribe virus, or a Whitewater Arroyo virus. In some embodiments, the Arenavirus is a Junin virus, a Machupo virus, a Sabia virus, or a Tacaribe virus. In some embodiments, the Arenavirus is JUNV strain Candid#1. In some embodiments, the Arenavirus is MACV strain Carvallo. In some embodiments, the Arenavirus is MACV strain Chicava. In some embodiments, the Arenavirus and the IGR Deletion Arenavirus are of the same viral species. In some embodiments, the Arenavirus and the IGR Deletion Arenavirus are of different viral species. In some embodiments, the Arenavirus is an OW Arenavirus and the IGR Deletion Arenavirus is a NW Arenavirus. In some embodiments, the Arenavirus is a NW Arenavirus and the IGR Deletion Arenavirus is an OW Arenavirus. In some embodiments, the Arenavirus and the IGR Deletion Arenavirus are each independently selected from the group consisting of Lassa virus, Lujo virus, Lymphocytic choriomeningitis virus, Chapare virus, Guanarito virus, Junin virus, Machupo virus, Sabia virus, Tacaribe virus, and Whitewater Arroyo virus. In some embodiments, the Arenavirus is a Lassa virus, a Lujo virus, a Lymphocytic choriomeningitis virus, a Chapare virus, a Guanarito virus, a Junin virus, a Machupo virus, a Sabia virus, a Tacaribe virus, or a Whitewater Arroyo virus and the IGR Deletion Arenavirus is JUNV strain Candid#1, MACV strain Carvallo, or MACV strain Chicava, and has a portion of its intragenic region (IGR) of the L-segment deleted. In some embodiments, the Arenavirus is a Guanarito virus or a Junin virus and the IGR Deletion Arenavirus is a Machupo virus. In some embodiments, the one or more IGR Deletion Arenaviruses are administered in the form of a composition as described herein. In some embodiments, the administration is before, during, and/or after exposure to the Arenavirus. In some embodiments, the Arenavirus and the IGR Deletion Arenavirus are of different viral species. In some embodiments, the Arenavirus is a Guanarito virus or a Junin virus and the IGR Deletion Arenavirus is a Machupo virus. In some embodiments, the one or more IGR Deletion Arenaviruses elicits a protective immune response against the Arenavirus when administered to the subject.
In some embodiments, the present invention provides a method of eliciting an immunogenic response in a subject, which comprises administering to the subject an immunogenic amount of one or more IGR Deletion Arenaviruses, which have at least a portion of an intergenic region (IGR) of one or both genome segments of an Arenavirus. In some embodiments, the portion of the IGR is deleted using recombinant DNA methods known in the art. In some embodiments, the genome segment is the L-segment. In some embodiments, the genome segment is the S-segment. In some embodiments, at least about 15-50 nucleotide bases of the IGR are deleted. In some embodiments, at least about 20-45 nucleotide bases of the IGR are deleted. In some embodiments, at least about 25-40 nucleotide bases of the IGR are deleted. In some embodiments, at least about 30-35 nucleotide bases of the IGR are deleted. In some embodiments, at least about 20%, preferably at least about 30%, more preferably at least about 40%, even more preferably at least about 50%, and most preferably about 60% of the nucleotide bases of the IGR are deleted. In some embodiments, the native structure of the hairpin loop formed by the nucleotide sequence of the IGR of the Arenavirus is altered by the deletion of the nucleotide bases. In some embodiments, the Arenavirus is an OW Arenavirus. In some embodiments, the Arenavirus is a NW Arenavirus. In some embodiments, the Arenavirus is a Lassa virus, a Lujo virus, a Lymphocytic choriomeningitis virus, a Chapare virus, a Guanarito virus, a Junin virus, a Machupo virus, a Sabia virus, a Tacaribe virus, or a Whitewater Arroyo virus. In some embodiments, the Arenavirus is a Junin virus, a Machupo virus, a Sabia virus, or a Tacaribe virus. In some embodiments, the Arenavirus is JUNV strain Candid#1. In some embodiments, the Arenavirus is MACV strain Carvallo. In some embodiments, the Arenavirus is MACV strain Chicava. In some embodiments, the one or more IGR Deletion Arenaviruses are administered in the form of a composition as described herein. In some embodiments, the administration is before, during, and/or after exposure to an Arenavirus. In some embodiments, the Arenavirus and the IGR Deletion Arenavirus are of the same viral species. In some embodiments, the Arenavirus and the IGR Deletion Arenavirus are of different viral species. In some embodiments, the Arenavirus is an OW Arenavirus and the IGR Deletion Arenavirus is a NW Arenavirus. In some embodiments, the Arenavirus is a NW Arenavirus and the IGR Deletion Arenavirus is an OW Arenavirus. In some embodiments, the Arenavirus and the IGR Deletion Arenavirus are each independently selected from the group consisting of Lassa virus, Lujo virus, Lymphocytic choriomeningitis virus, Chapare virus, Guanarito virus, Junin virus, Machupo virus, Sabia virus, Tacaribe virus, and Whitewater Arroyo virus. In some embodiments, the Arenavirus is a Lassa virus, a Lujo virus, a Lymphocytic choriomeningitis virus, a Chapare virus, a Guanarito virus, a Junin virus, a Machupo virus, a Sabia virus, a Tacaribe virus, or a Whitewater Arroyo virus and the IGR Deletion Arenavirus is JUNV strain Candid#1, MACV strain Carvallo, or MACV strain Chicava, and has a portion of its intragenic region (IGR) of the L-segment deleted. In some embodiments, the Arenavirus is a Guanarito virus or a Junin virus and the IGR Deletion Arenavirus is a Machupo virus. In some embodiments, the one or more IGR Deletion Arenaviruses elicits an immune response against the Arenavirus when challenged therewith.
In some embodiments, the present invention is directed to the use of at least one IGR Deletion Arenavirus in the manufacture of a medicament for preventing, inhibiting, reducing, or treating an Arenavirus infection in a subject. In some embodiments, the present invention is directed to the use of at least one IGR Deletion Arenavirus for the manufacture of a medicament for preventing, inhibiting, reducing, or treating an Arenavirus infection, wherein the medicament is prepared to be administered in accordance with one or more of the dosage regimens as described herein. In some embodiments, the medicament comprises a therapeutically effective amount of the at least one IGR Deletion Arenavirus as described herein. In some embodiments, the medicament comprises a pharmaceutically acceptable carrier. In some embodiments, the medicament comprises an adjuvant.
In some embodiments, the subject of the methods of the present invention is a mammalian subject. In some embodiments, the subject is a human subject. In some embodiments, the subject is one who is in need of treatment with at least one IGR Deletion Arenavirus. As used herein, a subject who is in need of treatment with at least one IGR Deletion Arenavirus is one who has, will, or will likely be exposed to an Arenavirus.
In embodiments where the IGR Deletion Arenavirus is an Arenavirus that is MACV strain Carvallo that has a deletion of a portion of an intergenic region (IGR) of one or both of its genome segments, the IGR Deletion Arenavirus is not Car⁹¹.
Both the foregoing general description and the following detailed description are exemplary and explanatory only and are intended to provide further explanation of the invention as claimed. The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute part of this specification, illustrate several embodiments of the invention, and together with the description explain the principles of the invention.

DESCRIPTION OF THE DRAWINGS

Both the foregoing general description and the following detailed description are exemplary and explanatory only and are intended to provide further explanation of the invention as claimed. The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute part of this specification, illustrate several embodiments of the invention, and together with the description explain the principles of the invention.

This invention is further understood by reference to the drawings wherein:

FIG. 1, FIG. 2, and FIG. 3 are graphs showing the weight loss, change in temperature, and PsVNA80 titers in Hartley guinea pigs after infection with MACV strain Carvallo. FIG. 1: Guinea pigs were infected with 2000 pfu MACV strains Car⁹¹and weights based on Day 0 starting weight were graphed. FIG. 2: Percent change in temperature relative to Day 0 was plotted. FIG. 3: Serum from guinea pigs was incubated with VSVΔG particles pseudotyped with MACV strain Carvallo glycoprotein and the PsVNA80 titers were graphed.

FIG. 4, FIG. 5, and FIG. 6 are graphs summarizing the results of in vitro characterization assays of Car⁹¹, Car⁶⁸, and Chic. FIG. 4: Particle-to-pfu ratios for each of the indicated MACV strains were determined using a ViroCyte system. Four virus preparations for each virus were tested in triplicate. The GMTs are indicated by the solid line. FIG. 5: Indicated cells were infected with Car⁹¹(circles), Car⁶⁸(squares) or Chic (triangles) and replication quantitated at 24, 48, and 72 hpi by plaque assay. All samples were titered in duplicate and the mean+/−SD were graphed. FIG. 6: Anti-glycoprotein antisera from four rabbits vaccinated with MACV strain Carvallo GPc [29] was serially diluted and incubated with Car⁶⁸, Car⁹¹, and Chic. PRNT50 and PRNT80 titers were plotted.

FIG. 7, FIG. 8, and FIG. 9 summarize the sequence analyses of MACV strain Car⁹¹and Car⁶⁸. FIG. 7: Predicted L-segment amino acid changes between Car⁹¹and Car⁶⁸. FIG. 8: Nucleotide line up of Car⁹¹(SEQ ID NO: 1) and Car⁶⁸(SEQ ID NO: 2). The underlined region denotes the deleted region. Also underlined is the single nucleotide change at position 399. FIG. 9 schematically shows the predicted hairpin tertiary structure of the Car⁹¹(SEQ ID NO: 3) and Car⁶⁸(SEQ ID NO: 4) L-segment IGRs.

FIG. 10, FIG. 11, FIG. 12, FIG. 13, FIG. 14 and FIG. 15 summarize infection of Hartley guinea pigs with MACV strain Car⁶⁸, Car⁹¹, and Chic. FIG. 10: Guinea pigs were i.p. infected with 1000 pfu of MACV strains Car⁹¹, Car⁶⁸, and Chic. Survival was monitored for 30 days post-infection. Percent weight loss for each group (FIG. 11) and individual guinea pigs (FIG. 12, FIG. 13, and FIG. 14) were plotted based on Day 0 starting weight. Animals succumbing to infection are shown in solid points in FIG. 13, and all points in FIG. 14. FIG. 15: Group temperatures were plotted. Normal temperature values are shown as a solid grey area.

FIG. 16, FIG. 17, and FIG. 18 are graphs summarizing the results of viremia and antibody assays on serum from infected guinea pigs. FIG. 16: Serum viremia from animals succumbing to disease was determined on Vero cell monolayers. The solid line represents the GMT values. FIG. 17: Antibody binding titers were determined by incubating sera from Car⁹¹infected animals with PsVs pseudotyped with GPc from strain Carvallo. Antiserum samples were serially diluted prior to incubation. The dashed line denotes the limit of detection. The solid line represents the GMT values. FIG. 18: Titers of neutralizing antibody in Car⁹¹infected animals was determined and PRNT50 and PRNT80 titers were plotted. The dashed line denotes the limit of detection. The solid line represents the GMT values.

FIG. 19, FIG. 20, and FIG. 21 are graphs showing the protective efficacy of MACV strain Car⁹¹against lethal GTOV challenge in guinea pigs. FIG. 19: Survival plot of guinea pigs were infected by the i.p. route with 2000 pfu GTOV. Survival was plotted for 30 day post-infection. FIG. 20: Percent loss from starting weight was plotted for each group as described above. FIG. 21: Temperature was monitored as in FIG. 16 to FIG. 18.

FIG. 22, FIG. 23, and FIG. 24 graphically summarize the binding and neutralizing antibody responses in guinea pigs infected with GTOV. FIG. 22: Antibody binding titers were determined by coating 96-well plates with the indicated PsVs and incubating them with serially diluted antiserum samples from before GTOV challenge (circles/PRE) or after GTOV challenge (squares/POST). The dashed line denotes the limits of detection. The red circle (on the horizontal line, the first dot in the first graph, the second dot in the second graph, and the only dot in the third graph) denotes the single animal (Animal #4) that died from infection. FIG. 23: PRNT80 titers against MACV prior to and after challenge with GTOV. PRNT50 titers were determined as above. FIG. 24: PRNT50 titers against JUNV, MACV, and GTOV were determined as in FIG. 4 to FIG. 6. Titers were determined as described above. The dashed line indicates the limits of detection.

DETAILED DESCRIPTION OF THE INVENTION

MACV strain Carvallo is the prototypical MACV strain first isolated in 1963 [26, 27]. Several studies have found strain Carvallo is lethal in guinea pigs, with lethality upwards of >60% [20, 28]. Previously, it was reported that a MACV strain Carvallo variant (Car⁹¹) does not cause lethal disease in guinea pigs [29]. To reconcile this discrepancy, as disclosed herein, the attenuated strain Car⁹¹was compared with an earlier isolate of strain Carvallo, Car⁶⁸. The virulence of Car⁹¹and Car⁶⁸was examined in guinea pigs, the classic small animal model for Arenavirus [27]. Virulence was also compared to MACV strain Chicava (Chic), which was recently reported to cause lethal disease in guinea pigs [30]. The genetic mechanism behind the attenuation of Car⁹¹was investigated along with its ability to function as a pan-vaccine against NW Arenaviruses.
As disclosed herein, Car⁹¹was discovered to have a deletion of 35 nucleotides in the intragenic region (IGR) of the L-segment of the bisegmented genome. This mutation, which appears to have occurred during passage in culture cells as the originally isolated virus taken from an infected person in the 1960s does not contain this mutation, impacts replication kinetics. Contrary to the wildtype (natural) strain of Carvallo, Car⁹¹does not cause a lethal infection in infected guinea pigs. Also, as disclosed herein, Car⁹¹produces antibodies, including neutralizing antibodies that do not cross-neutralize other related NW Arenaviruses, in animals. Surprisingly, it was found that guinea pigs initially challenged with Car⁹¹are protected against infection by the Guanarito virus, a distantly related NW Arenavirus. That is, a deletion in the L-segment IGR of a given Arenavirus results in an attenuated virus that provides protective immunity against other Arenaviruses that are distantly related to the given Arenavirus.
Therefore, in some embodiments, the present invention provides a method of making an attenuated form of an Arenavirus, which comprises deleting at least a portion of an IGR of one or both genome segments of the Arenavirus. In some embodiments, recombinant DNA techniques are used to delete the portion of the IGR. In some embodiments, the genome segment is the L-segment. In some embodiments, the genome segment is the S-segment. In some embodiments, at least about 15-50 nucleotide bases of the IGR are deleted. In some embodiments, at least about 20-45 nucleotide bases of the IGR are deleted. In some embodiments, at least about 25-40 nucleotide bases of the IGR are deleted. In some embodiments, at least about 30-35 nucleotide bases of the IGR are deleted. In some embodiments, at least about 20%, preferably at least about 30%, more preferably at least about 40%, even more preferably at least about 50%, and most preferably about 60% of the nucleotide bases of the IGR are deleted. In some embodiments, all the nucleotide bases of the IGR are deleted. In some embodiments, the native structure of the hairpin loop formed by the nucleotide sequence of the IGR of the Arenavirus is altered by the deletion of the nucleotide bases. In some embodiments, the Arenavirus is an OW Arenavirus. In some embodiments, the Arenavirus is a NW Arenavirus. In some embodiments, the Arenavirus is a Lassa virus, a Lujo virus, a Lymphocytic choriomeningitis virus, a Chapare virus, a Guanarito virus, a Junin virus, a Machupo virus, a Sabia virus, a Tacaribe virus, or a Whitewater Arroyo virus. In some embodiments, the Arenavirus is a Junin virus, a Machupo virus, a Sabia virus, or a Tacaribe virus. In some embodiments, the Arenavirus is JUNV strain Candid#1. In some embodiments, the Arenavirus is MACV strain Carvallo. In some embodiments, the Arenavirus is MACV strain Chicava.
In some embodiments, the present invention provides IGR Deletion Arenaviruses and compositions comprising one or more IGR Deletion Arenaviruses. As used herein, a “IGR Deletion Arenavirus” refers to an Arenavirus that has been attenuated by using recombinant DNA techniques to delete at least a portion of an IGR of one or both genome segments of the native form of the Arenavirus. In some embodiments, the genome segment is the L-segment. In some embodiments, the genome segment is the S-segment. In some embodiments, at least about 15-50 nucleotide bases of the IGR are deleted. In some embodiments, at least about 20-45 nucleotide bases of the IGR are deleted. In some embodiments, at least about 25-40 nucleotide bases of the IGR are deleted. In some embodiments, at least about 30-35 nucleotide bases of the IGR are deleted. In some embodiments, at least about 20%, preferably at least about 30%, more preferably at least about 40%, even more preferably at least about 50%, and most preferably about 60% of the nucleotide bases of the IGR are deleted. In some embodiments, the native structure of the hairpin loop formed by the nucleotide sequence of the IGR of the Arenavirus is altered by the deletion of the nucleotide bases. In some embodiments, the Arenavirus is an OW Arenavirus. In some embodiments, the Arenavirus is a NW Arenavirus. In some embodiments, the Arenavirus is a Lassa virus, a Lujo virus, a Lymphocytic choriomeningitis virus, a Chapare virus, a Guanarito virus, a Junin virus, a Machupo virus, a Sabia virus, a Tacaribe virus, or a Whitewater Arroyo virus. In some embodiments, the Arenavirus is a Junin virus, a Machupo virus, a Sabia virus, or a Tacaribe virus. In some embodiments, the Arenavirus is JUNV strain Candid#1. In some embodiments, the Arenavirus is MACV strain Carvallo. In some embodiments, the Arenavirus is MACV strain Chicava.
In some embodiments, the present invention provides a method of treating, inhibiting, or reducing an Arenavirus infection or a disease caused by an Arenavirus in a subject, which comprises administering to the subject an effective amount of one or more IGR Deletion Arenaviruses. As used herein, an “Arenavirus infection” refers to an infection by one or more viruses belonging to the family Arenaviridae. Diseases caused by Arenavirus infections include hemorrhagic fevers, Lassa fever, Lymphocytic choriomeningitis, and Influenza-like illness. Hemorrhagic fevers include Argentine hemorrhagic fever, Bolivian hemorrhagic fever, Venezuelan hemorrhagic fever, and Brazilian hemorrhagic fever. In some embodiments, the effective amount is a therapeutically effective amount. In some embodiments, the effective amount is an immunogenic amount. In some embodiments, the one or more IGR Deletion Arenaviruses is administered before, during, and/or after exposure to the Arenavirus. In some embodiments, the Arenavirus and the IGR Deletion Arenavirus are of the same viral species. In some embodiments, the Arenavirus and the IGR Deletion Arenavirus are of different viral species. In some embodiments, the Arenavirus is an OW Arenavirus and the IGR Deletion Arenavirus is a NW Arenavirus. In some embodiments, the Arenavirus is a NW Arenavirus and the IGR Deletion Arenavirus is an OW Arenavirus. In some embodiments, the Arenavirus and the IGR Deletion Arenavirus are each independently selected from the group consisting of Lassa virus, Lujo virus, Lymphocytic choriomeningitis virus, Chapare virus, Guanarito virus, Junin virus, Machupo virus, Sabia virus, Tacaribe virus, and Whitewater Arroyo virus. In some embodiments, the Arenavirus is a Lassa virus, a Lujo virus, a Lymphocytic choriomeningitis virus, a Chapare virus, a Guanarito virus, a Junin virus, a Machupo virus, a Sabia virus, a Tacaribe virus, or a Whitewater Arroyo virus and the IGR Deletion Arenavirus is JUNV strain Candid#1, MACV strain Carvallo, or MACV strain Chicava, and has a portion of its intragenic region (IGR) of the L-segment deleted. In some embodiments, the Arenavirus is a Guanarito virus or a Junin virus and the IGR Deletion Arenavirus is a Machupo virus.
In some embodiments, the present invention provides a method of immunizing a subject against an Arenavirus, which comprises administering to the subject an immunogenic amount of one or more IGR Deletion Arenaviruses. In some embodiments, the one or more IGR Deletion Arenaviruses is administered before, during, and/or after exposure to the Arenavirus. In some embodiments, the Arenavirus and the IGR Deletion Arenavirus are of the same viral species. In some embodiments, the Arenavirus and the IGR Deletion Arenavirus are of different viral species. In some embodiments, the Arenavirus is an OW Arenavirus and the IGR Deletion Arenavirus is a NW Arenavirus. In some embodiments, the Arenavirus is a NW Arenavirus and the IGR Deletion Arenavirus is an OW Arenavirus. In some embodiments, the Arenavirus and the IGR Deletion Arenavirus are each independently selected from the group consisting of Lassa virus, Lujo virus, Lymphocytic choriomeningitis virus, Chapare virus, Guanarito virus, Junin virus, Machupo virus, Sabia virus, Tacaribe virus, and Whitewater Arroyo virus. In some embodiments, the Arenavirus is a Lassa virus, a Lujo virus, a Lymphocytic choriomeningitis virus, a Chapare virus, a Guanarito virus, a Junin virus, a Machupo virus, a Sabia virus, a Tacaribe virus, or a Whitewater Arroyo virus and the IGR Deletion Arenavirus is JUNV strain Candid#1, MACV strain Carvallo, or MACV strain Chicava, and has a portion of its intragenic region (IGR) of the L-segment deleted. In some embodiments, the Arenavirus is a Guanarito virus or a Junin virus and the IGR Deletion Arenavirus is a Machupo virus. In some embodiments, the immunogenic amount results in a protective immune response against the Arenavirus.
Compositions of the present invention, including pharmaceutical compositions and vaccines, include one or more IGR Deletion Arenaviruses as described herein. The term “pharmaceutical composition” refers to a composition suitable for pharmaceutical use in a subject. A pharmaceutical composition generally comprises an effective amount of an active agent, e.g., one or more IGR Deletion Arenaviruses according to the present invention, and a pharmaceutically acceptable carrier. The term “effective amount” refers to a dosage or amount sufficient to produce a desired result. The desired result may comprise an objective or subjective improvement in the recipient of the dosage or amount, e.g., long-term survival, effective prevention of a disease state, and the like. Pharmaceutical compositions according to the present invention may further include one or more supplementary agents. Examples of suitable supplementary agents include ribavirin, antivirals, and the like.
One or more IGR Deletion Arenaviruses according to the present invention may be administered, preferably in the form of pharmaceutical compositions, to a subject. Preferably the subject is mammalian, more preferably, the subject is human. Preferred pharmaceutical compositions are those comprising, consisting essentially of, or consisting of at least one IGR Deletion Arenavirus in a therapeutically effective amount or an immunogenic amount, and a pharmaceutically acceptable vehicle.
Vaccines according to the present invention provide a protective immune response when administered to a subject. As used herein, a “vaccine” according to the present invention is a pharmaceutical composition that comprises an immunogenic amount of at least one IGR Deletion Arenavirus and provides a protective immune response when administered to a subject. The protective immune response may be complete or partial, e.g., a reduction in symptoms as compared with an unvaccinated subject.
As used herein, an “immunogenic amount” is an amount that is sufficient to elicit an immune response in a subject and depends on a variety of factors such as the immunogenicity of the given IGR Deletion Arenavirus, the degree of infection by or exposure to an Arenavirus, the manner of administration, the general state of health of the subject, and the like. The typical immunogenic amounts for initial and boosting immunizations for therapeutic or prophylactic administration may range from about 10¹to about 10⁷plaque forming units (pfu) or equivalent TCID₅₀. In some embodiments, the immunogenic amount is about 10²to about 10⁷plaque forming units (pfu) or equivalent TCID₅₀. In some embodiments, the immunogenic amount is about 10³to about 10⁷plaque forming units (pfu) or equivalent TCID₅₀. The vaccine initial vaccine and booster vaccination may be the same dosage or different. Similarly, for prophylactic and therapeutic doses, the amount of attenuated virus used may be different. For example, a therapeutic dose of a given IGR Deletion Arenavirus may be 10⁷pfu or equivalent TCID₅₀, while the therapeutic dose of a different IGR Deletion Arenavirus may be 1000 pfu or equivalent TCID₅₀. Examples of suitable immunization protocols include an initial immunization injection (time 0), followed by booster injections (if needed) at 4, and/or 8 weeks, which these initial immunization injections may be followed by further booster injections at 1 or 2 years if needed.
As used herein, a “therapeutically effective amount” refers to an amount that may be used to treat, prevent, or inhibit an Arenavirus infection in a subject as compared to a control. Again, the skilled artisan will appreciate that certain factors may influence the amount required to effectively treat a subject, including the degree of infection by or exposure to an Arenavirus, previous treatments, the general health and age of the subject, and the like. Nevertheless, therapeutically effective amounts may be readily determined by methods in the art. It should be noted that treatment of a subject with a therapeutically effective amount or an immunogenic amount may be administered as a single dose or as a series of several doses. The dosages used for treatment may increase or decrease over the course of a given treatment. Optimal dosages for a given set of conditions may be ascertained by those skilled in the art using dosage-determination tests and/or diagnostic assays in the art. Dosage-determination tests and/or diagnostic assays may be used to monitor and adjust dosages over the course of treatment.
The compositions of the present invention may include an adjuvant. As used herein, an “adjuvant” refers to any substance which, when administered in conjunction with (e.g., before, during, or after) a pharmaceutically active agent, such as a IGR Deletion Arenavirus according to the present invention, aids the pharmaceutically active agent in its mechanism of action. Thus, an adjuvant in a vaccine according to the present invention is a substance that aids the at least one IGR Deletion Arenavirus in eliciting an immune response. Suitable adjuvant include incomplete Freund's adjuvant, alum, aluminum phosphate, aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, nor-MDP), N-acetylmuramyl-Lalanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipa-lmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, MTP-PE), and RIBI, which comprise three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (NPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion. The effectiveness of an adjuvant may be determined by methods in the art.
Pharmaceutical compositions of the present invention may be formulated for the intended route of delivery, including intravenous, intramuscular, intra peritoneal, subcutaneous, intraocular, intrathecal, intraarticular, intrasynovial, cisternal, intrahepatic, intralesional injection, intracranial injection, infusion, and/or inhaled routes of administration using methods known in the art. Pharmaceutical compositions according to the present invention may include one or more of the following: pH buffered solutions, adjuvants (e.g., preservatives, wetting agents, emulsifying agents, and dispersing agents), liposomal formulations, nanoparticles, dispersions, suspensions, or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions. The compositions and formulations of the present invention may be optimized for increased stability and efficacy using methods in the art. See, e.g., Carra et al. (2007) Vaccine 25:4149-4158, which is herein incorporated by reference.
The compositions of the present invention may be administered to a subject by any suitable route including oral, transdermal, subcutaneous, intranasal, inhalation, intramuscular, and intravascular administration. It will be appreciated that the preferred route of administration and pharmaceutical formulation will vary with the condition and age of the subject, the nature of the condition to be treated, the therapeutic effect desired, and the particular IGR Deletion Arenavirus used.
As used herein, a “pharmaceutically acceptable vehicle”, “pharmaceutically acceptable carrier”, and “pharmaceutically acceptable excipient” are used interchangeably and refer to solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration and comply with the applicable standards and regulations, e.g., the pharmacopeial standards set forth in the United States Pharmacopeia and the National Formulary (USP-NF) book, for pharmaceutical administration. Thus, for example, unsterile water is excluded as a pharmaceutically acceptable carrier for, at least, intravenous administration. In some embodiments, pharmaceutically acceptable vehicles, carriers, and excipients refer to a human-made substance that is of a purity level that is acceptable for administration to humans, e.g., sterile water that is acceptable for intravenous administration. Pharmaceutically acceptable vehicles include those known in the art. See, e.g., Remington: The Science and Practice of Pharmacy. 20^thed. (2000) Lippincott Williams & Wilkins. Baltimore, Md., which is herein incorporated by reference.
The pharmaceutical compositions of the present invention may be provided in dosage unit forms. As used herein, a “dosage unit form” refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of the one or more IGR Deletion Arenavirus calculated to produce the desired therapeutic effect in association with the required pharmaceutically acceptable carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the given IGR Deletion Arenavirus and desired therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
Toxicity and therapeutic efficacy of IGR Deletion Arenaviruses according to the instant invention and compositions thereof can be determined using cell cultures and/or experimental animals and pharmaceutical procedures in the art. For example, one may determine the lethal dose, LC₅₀(the dose expressed as concentration×exposure time that is lethal to 50% of the population) or the LD₅₀(the dose lethal to 50% of the population), and the ED₅₀(the dose therapeutically effective in 50% of the population) by methods in the art. The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀. IGR Deletion Arenaviruses which exhibit large therapeutic indices are preferred. While IGR Deletion Arenaviruses that result in toxic side-effects may be used, care should be taken to design a delivery system that targets such compounds to the site of treatment to minimize potential damage to uninfected cells and, thereby, reduce side-effects.
The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosages for use in humans. Preferred dosages provide a range of circulating concentrations that include the ED₅₀with little or no toxicity. The dosage may vary depending upon the dosage form employed and the route of administration utilized. Therapeutically effective amounts and dosages of one or more IGR Deletion Arenaviruses according to the present invention can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC₅₀(i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography. Additionally, a dosage suitable for a given subject can be determined by an attending physician or qualified medical practitioner, based on various clinical factors.
In some embodiments, the present invention is directed to kits which comprise one or more IGR Deletion Arenaviruses, optionally in a composition or in combination with one or more supplementary agents, packaged together with one or more reagents or drug delivery devices for preventing, inhibiting, reducing, or treating Arenavirus infection in a subject. Such kits include a carrier, package, or container that may be compartmentalized to receive one or more containers, such as vials, tubes, and the like. In some embodiments, the kits optionally include an identifying description or label or instructions relating to its use. In some embodiments, the kits comprise the one or more IGR Deletion Arenaviruses, optionally in one or more unit dosage forms, packaged together as a pack and/or in drug delivery device, e.g., a pre-filled syringe. In some embodiments, the kits include information prescribed by a governmental agency that regulates the manufacture, use, or sale of compounds and compositions according to the present invention.
The following examples are intended to illustrate but not to limit the invention.

Methods

Viruses and Cells

GTOV strain INH95551, MACV strain Chic, and two MACV strains of Carvallo from passages dated 1968 (Car⁶⁸) and 1991 (Car⁹¹) were propagated in Vero cell monolayers (ATCC CRL-1587) as previously reported [29]. All work with the viruses was performed in registered and certified biological safety level 4 (BSL4) containment environments. 239T cells, 104CL guinea pig fibroblasts (ATCC) were maintained in MEM or RPMI containing 10% heat-inactivated fetal bovine serum (FBS), 1% antibiotics (100 U/ml penicillin, 100 μg/ml of streptomycin, respectively. Human umbilical vein cells (HUVECs) (Lonza) were maintained in manufacture's medium.

Plaque Reduction and Neutralization Tests (PRNTs)

PRNTs were performed as previously described [31] using rabbit antiserum targeting MACV glycoprotein [29] or guinea pig serum (this study) serially diluted two-fold starting at 1:40. Percent neutralization was calculated relative to the number of plaques in the presence of negative control serum. Titers represent the reciprocal of the highest dilution resulting in a 50% reduction in the number of plaques. Data were plotted using Graphpad Prism software.

Growth Kinetics

Vero, 104CL, and HUVECs were seeded at a density of 1×10⁵cells per well in 24-well plates and infected at an MOI of 0.1 with the indicated viruses diluted in culture medium. Virus growth at 24, 48, and 72 hours was determined by plaque assay on Vero cell monolayers. All samples were run in duplicate and plotted as the mean+/−standard deviation (SD) using Graphpad Prism software.

Particle-to-PFU Ratio

Particle counts were determined with a Virocyt machine (Virocyt, Boulder, Colo.) using the manufacture's protocol. The particle-to-pfu ratios were determined by dividing particle counts by the amount of infectious virus. Four virus preparations per strain were used in the calculations.

Genome Sequencing

RNA was extracted from Trizol homogenates of MACV, converted to cDNA, and subjected to sequence-independent, single primer amplification (SISPA) [32]. The products of these reactions were used to generate libraries that were sequenced on an Illumina MiSeq. Sequencing reads were assembled using DNAStar SeqMan NGen.

Pseudovirion Neutralization Assay (PsVNA) and ELISA

The pseudovirion neutralization assay (PsVNA) used to detect Arenavirus neutralizing antibodies in sera has been described in detail elsewhere [29, 33]. Briefly, a vesicular stomatitis virus backbone with a luciferase reporter gene (PsV) was used to produce particles decorated with glycoproteins from MACV, JUNV, and GTOV. These particles were subsequently incubated with the indicated serially diluted sera in triplicate and the geometric mean titers (GMT) of PsVNA50 plotted. The pseudotyped VSV ELISA assay has been previously described in detail [29].

Challenge of Hartley Guinea Pigs

Female Hartley guinea pigs (300-400 g) were implanted with IPTT-3000 identification chips to monitor temperature (BMDS INC; Seaford, Del.). Animals were challenged with the indicated MACV strains (1,000 pfu) or GTOV (2000 pfu) diluted in a total volume of 0.5 ml PBS by intraperitoneal (i.p.) injection. Animals were weighed and monitored for fever. All animal studies were conducted in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals and adheres to principles state in the Guide for the Care and Use of Laboratory Animals, National Research Council, 1996. The facilities where this research was conducted are fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. Animals meeting criteria were humanly euthanized.

Statistical Analysis

Two-way ANOVA with the Bonferroni correction was used to determine the significance of weight loss and in vitro virus replication. Survival statistics utilized the log-rank test. The statistical significance of PRNTs was determined using the unpaired two-tailed Student's t test. Significance levels were set at a p value less than 0.05. All analyses were performed using Prism software.

Results

MACV Strain Carvallo is not Lethal in Hartley Guinea Pigs

MACV strain Car⁹¹was tested for its ability to produce an acute infection in Hartley guinea pigs. Six guinea pigs were infected with 2000 pfu of strain Carvallo and monitored for weight (FIG. 1) and temperature (FIG. 2) for 28 days post-infection. All animals gained weight over the course of the study and by Day 28 was >30% higher than the starting weights. Animals did not exhibit signs of infection, such as lethargy and no animal produced a fever. Serum from infected animals was tested for MACV neutralizing antibody using VSV particles pseudotyped with the MACV strain Carvallo glycoprotein molecules. Each guinea pig produced a neutralizing antibody response with the PsVNA80 GMT of 177 (FIG. 3). These results indicated that contrary to published reports [20, 28], MACV strain Carvallo failed to produce acute disease in infected guinea pigs. However, the virus elicited a neutralizing antibody response in inoculated animals.

In Vitro Characterization of Two MACV Strain Carvallo Passages and Strain Chic

Because the failure of Car⁹¹to cause lethality was surprising, Car⁶⁸, which is another stock of virus derived from an early passage of strain Carvallo (1968), was produced. Plaque formation on Vero cells was no unique between Car⁹¹and Car⁶⁸(data not shown). The particle-to-pfu ratio of the isolate was compared to Car⁶⁸and Chic to determine if Car⁹¹had more defective particles (FIG. 4). There were significant differences in the particle to pfu ratios between Car⁹¹, Car⁶⁸, and Chic. Car⁹¹had the highest particle count with a GMT of 369, compared to Car⁶⁸and Chic whose GMT ratios were 26 and 13, respectively.
The growth kinetics of all three strains was investigated in Vero cells, 104CL guinea pig fibroblasts, and HUVECs. Cells were infected with Car⁹¹, Car⁶⁸, or Chic and replication was assayed at 24, 48, and 72 hours post-infection (hpi) (FIG. 5). After 24 hours, Car⁶⁸grew to the highest levels in both Vero and HUVEC. The 24 hour growth in 104CL cells was markedly lower than Vero and HUVECs with values for all three viruses <1×10³pfu/ml, however growth of Car⁹¹was the lowest. At 48 hpi, Car⁹¹replication was still reduced compared to the other viruses in 104Cl and Vero cells, however in HUVECs Car⁹¹and Car⁶⁸had similar titers. Overall, Strain Chic grew to the highest levels in Vero and HUVECs and after 72 hours growth titers were 1.5×10⁷pfu/ml and 1.1×10⁶pfu/ml, respectively. The growth between Car⁶⁸(4.45×10⁵pfu/ml) and Chic (4.15×10⁵pfu/ml) was similar in 104CL cells. After 72 hours strain Car⁹¹titers were the lowest in all cell types with growth at 8.33×10⁵pfu/ml (Vero cells), 3.0×10⁵pfu/ml (HUVECs), and 1.2×10⁶pfu/ml (104CL). These titers were several fold lower than Car⁶⁸and Chic. Growth differences between Car⁹¹and Car⁶⁸were statistically significant (two-way ANOVA; p >0.05) at 72 hours (HUVECs and 104CL cells), but not in Vero cells. Growth titers were also significant between Car⁹¹and Chic at 72 hpi in all cell types. Together these findings indicate that MAV strain Car⁹¹is not as efficient at replication compared to other strains of MACV, including an older isolate of Carvallo from 1968.
There are no serotypes among the known human pathogenic NW Arenaviruses, however antibody neutralizing titers are typically multi-fold higher against homologous virus compared to heterologous strains [25, 34]. Therefore, the PRNTs of neutralizing antisera produced in rabbits was determined using plasmid DNA encoding the canonical MACV strain GPc gene to examined if Car⁹¹and Car⁶⁸are antigenically distinct [29]. The ability of the antisera to neutralize the heterologous strain Chic was also evaluated. As shown in FIG. 4 to FIG. 6, there was less than a 2-fold difference between Car⁹¹and Car⁶⁸PRNT50 and PRNT80 titers (FIG. 6). These titers did not reach a level of statistical difference (Car⁹¹versus Car⁶⁸; T-test; PRNT50 p=0.3436 and PRNT80 p=0.9130). In contrast, titers against heterologous virus strain Chic were markedly reduced and exhibiting about 10-fold reduction compared to Car⁹¹and Car⁶⁸. The PRNT50 values were statistical significant (T-test; p=0.0481), but not the PRNT80 values (p=0.1168). Thus, neutralizing titers against Car⁹¹and Car⁶⁸are indistinguishable suggesting they are highly serologically related.

MACV Strain Car⁹¹is Missing a Region of the L-Segment IGR

The genomes of Car⁹¹and Car⁶⁸were sequenced. Sequencing revealed five changes between strains Car⁶⁸and Car⁹¹(FIG. 7). Three changes resulted in silent mutations undisruptive to the protein sequence. Another change at position 399 results in a change in the IGR from C→T. This latter mutation matched the reference strain Carvallo sequence. In addition, a 35 nucleotide (nt) deletion in the IGR of strain Car⁹¹was identified (FIG. 8). No changes in the S segment were identified between Car⁹¹and Car⁶⁸. These findings demonstrated that Car⁹¹is missing a significant part of the L-segment IGR whereas these bases are present in the earlier passaged Car⁶⁸. This deletion resulted in a predicted IGR structure with ΔG value of −20.2 kcal/mol compared to 54.3 kcal/mol of Car⁶⁸(FIG. 9). Thus the Car⁶⁸structure is predicted to be more thermodynamically stable compared to Car⁹¹by 2.7-fold.
MACV Strain Car⁶⁸and Chic are Lethal in Guinea Pigs while Strain Car⁹¹is Attenuated
The virulence of strain Car⁶⁸was examined in Hartley guinea pigs to determine if, contrary to Car⁹¹, this isolate could produce acute disease. Animals were also infected with Chic, a strain known to cause lethal disease [30]. Groups of eight animals were infected with the indicated strains and survival, weight, and fever were monitored for 30 days (FIG. 10). All animals infected with strain Chic begin to lose weight between days 8-20 (FIG. 11, FIG. 12, FIG. 13, and FIG. 14), but only one animal developed high fever (>41.0° C.) (FIG. 15). Chic infected animals succumbed to infection by Day 24. Similarly, animals infected with Car⁶⁸displayed weight loss beginning between days 9 and 21, but none of the animals developed high fever (>41.0° C.). Car⁶⁸also produced a lethal disease in guinea pigs, however three animals survived infection (about 63% mortality rate). Distinct from Chic, three Car⁶⁸-infected animals developed paralysis starting with the hind-limb. These animals were euthanized on Day 21. The three surviving Car⁶⁸animals began to rapidly increase in weight after a period of weight loss, and by Day 30 they exceeded their starting with by about 3-20%. The maximum tolerated dose (MTD) for Car⁶⁸and Chic was 23.5 and 22, respectively. As expected, animals infected with Car⁹¹survived infection without displaying signs of disease (FIG. 10). Survival differences between Car⁶⁸and Chic were not significant (log-rank; p=0.1331) however the survival of Car⁹¹versus Car⁶⁸were highly significant (log-rank; p=0.0082). Additionally, weight loss between Car⁶⁸and Chic were also significant compared to Car⁹¹for several days (Two-way ANOVA: p<0.05). Viremia was undetected in any animal surviving challenge in the Car91 infected group. Viremia was detected in all four strain Chic infected animals that were euthanized due to disease severity with a GMT titer of 1088 pfu/ml (FIG. 16). Only one Car⁶⁸animal had detectable viremia with a titer of 166.0 pfu/ml. These results demonstrate that contrary to Car⁹¹, Car⁶⁸is lethal in guinea pigs. Consistent with previous reports [30], strain Chic was also found to be highly lethal in the guinea pig model.
The serum from Car91-challenged guinea pigs was evaluated for the presence of binding and neutralization antibodies 30 days post-challenge. ELISA titers were determined using VSVΔG particles pseudotyped with glycoproteins from the MACV strain Carvallo as antigen. Six of eight guinea pigs had detectable antibodies against MACV glycoprotein with Log₁₀GMT titers of 2.8 (FIG. 17). MACV neutralizing antibody was detected in all but two infected animals with PRNT50 and PRNT80 GMTs of 269 and 59.5, respectively (FIG. 18). These findings further demonstrated that Car⁹¹can consistently produce humoral responses in infected animals without disease.

MACV Strain Car⁹¹Protects Guinea Pigs Against Lethal Infection by GTOV

Because strain Car⁹¹was highly attenuated in guinea pigs but produced detectable immune responses, it was hypothesized that it might function as a live-attenuated vaccine. To this end, the ability of Car⁹¹to protect guinea pigs against GTOV, a distantly related NW Arenavirus [35]. Guinea pigs were challenged with GTOV 45 days after exposure to MACV (FIG. 19, FIG. 20, and FIG. 21). As a control for acute infection, a group of six weight-matched naïve guinea pigs were also infected. Animals were monitored for survival, weight loss, and fever over 25 days (FIG. 19, FIG. 20, and FIG. 21). Consistent with previous work [29, 36], all control animals began to lose weight starting around Day 6 with concomitant fever. All control animals succumbed to infection with a MTD of 16 days. All but one animal previously exposed to Car⁹¹survived infection. The single non-survivor succumbed to disease on Day 17 after a period of weight loss and mild fever (about 40.3° C.).
The presence of binding antibody responses against MACV, JUNV, and GTOV were evaluated by ELISA using sera collected prior to and after GTOV challenge (FIG. 22). Prior to GTOV challenge, six of eight animals infected with MACV strain Car⁹¹had detectable antibodies against MACV with a log₁₀GMT of 2.5. These responses increased following GTOV challenge to 3.6, but this was not significant (T-test; p=0.1461). Except for two animals, antibody titers against GTOV prior to GTOV challenge were below detection. However, antibody titers rose significantly (T-test; p=0.0002) after GTOV challenge to a GMTlog₁₀GMT of 2.5. Antibody titers against JUNV were also detected prior to GTOV challenge in all but two animals and these responses increased to statistically significant levels (T-test; p=0.0242) with logic) GMT of 3.0 after GTOV challenge. Animal #4, which succumbed to GTOV infection even after receiving Car⁹¹had undetectable ELISA tiers against MACV, GTOV, and JUNV.
The PRNT titers against MACV significantly increased subsequent to GTOV challenge, with PRNT80 GMTs rising from 59 to 320 (T-test; p=0.0112) (FIG. 23). Additionally, animal #1 with the lowest PRNT against MACV had the lowest IgG titer against MACV, GTOV, and JUNV before and after GTOV challenge. Despite the presence of MACV, GTOV, and JUNV antibody in all animals surviving GTOV challenge, neutralizing activity against GTOV or JUNV was undetected (FIG. 24). These findings demonstrated that MACV strain Car⁹¹protects animals against GTOV.

REFERENCES

The following references are herein incorporated by reference in their entirety:

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The examples herein are intended to illustrate but not to limit the invention.
All scientific and technical terms used in this application have meanings commonly used in the art unless otherwise specified.
As used herein, the terms “subject”, “patient”, and “individual” are used interchangeably to refer to humans and non-human animals. The term “non-human animal” includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, horses, sheep, dogs, cows, pigs, chickens, and other veterinary subjects and test animals. In some embodiments of the present invention, the subject is a mammal. In some embodiments of the present invention, the subject is a human.
The use of the singular can include the plural unless specifically stated otherwise. As used in the specification and the appended claims, the singular forms “a”, “an”, and “the” can include plural referents unless the context clearly dictates otherwise. As used herein, “and/or” means “and” or “or”. For example, “A and/or B” means “A, B, or both A and B” and “A, B, C, and/or D” means “A, B, C, D, or a combination thereof” and said “combination thereof” means any subset of A, B, C, and D, for example, a single member subset (e.g., A or B or C or D), a two-member subset (e.g., A and B; A and C; etc.), or a three-member subset (e.g., A, B, and C; or A, B, and D; etc.), or all four members (e.g., A, B, C, and D).
As used herein, the term “comprising” is used in its conventional sense to indicate that the given composition (or method) may include other ingredients (or steps). As used herein, the term “consisting of” is used in its conventional sense to indicate that the given composition (or method) may not include any additional ingredients (or steps). As used herein, the phrase “consists essentially of” indicates that the given composition (or method) may include other ingredients (or steps) so long as the additional ingredients (or steps) do not materially change the biological and/or chemical activity (or results) of the specified ingredients (or steps).
The phrase “comprises, consists essentially of”, or consists of is used as a tool to avoid excess page and translation fees and means that in some embodiments the given thing at issue comprises something, and in some embodiments the given thing at issue consists of something. For example, the sentence “In some embodiments, the composition comprises, consists essentially of, or consists of A” is to be interpreted as if written as the following two separate sentences: “In some embodiments, the composition comprises A. In some embodiments, the composition consists essentially of A. In some embodiments, the composition consists of A.” Similarly, a sentence reciting a string of alternates is to be interpreted as if a string of sentences were provided such that each given alternate was provided in a sentence by itself. For example, the sentence “In some embodiments, the composition comprises A, B, or C” is to be interpreted as if written as the following three separate sentences: “In some embodiments, the composition comprises A. In some embodiments, the composition comprises B. In some embodiments, the composition comprises C.”
To the extent necessary to understand or complete the disclosure of the present invention, all publications, patents, and patent applications mentioned herein are expressly incorporated by reference therein to the same extent as though each were individually so incorporated.
Having thus described exemplary embodiments of the present invention, it should be noted by those skilled in the art that the within disclosures are exemplary only and that various other alternatives, adaptations, and modifications may be made within the scope of the present invention. Accordingly, the present invention is not limited to the specific embodiments as illustrated herein, but is only limited by the following claims.

Claims

1. A method of making an IGR Deletion Arenavirus, which comprises deleting at least a portion of an intergenic region (IGR) of one or both genome segments of an Arenavirus, wherein the IGR Deletion Arenavirus is not Car91.

2. The method according to claim 1, wherein the genome segment is the L-segment.

3. The method according to claim 1, wherein at least about 20% of the nucleotide bases of the IGR are deleted.

4. The method according to claim 1, wherein at least about 15-50 nucleotide bases of the IGR are deleted.

5. The method according to claim 1, wherein the IGR has a hairpin loop and deleting the portion of the IGR alters the structure of the hairpin loop.

6. The method according to claim 1, wherein the Arenavirus is a Lassa virus, a Lujo virus, a Lymphocytic choriomeningitis virus, a Chapare virus, a Guanarito virus, a Junin virus, a Machupo virus, a Sabia virus, a Tacaribe virus, or a Whitewater Arroyo virus.

7. The method according to claim 1, wherein the Arenavirus is a Junin virus, a Machupo virus, a Sabia virus, or a Tacaribe virus.

8. The method according to claim 1, wherein the Arenavirus is a Machupo virus.

9. The method according to claim 1, wherein the Arenavirus is JUNV strain Candid#1, MACV strain Carvallo, or MACV strain Chicava.

10. The method according to claim 1, wherein the IGR Deletion Arenavirus is an attenuated form of the Arenavirus.

11. (canceled)

12. A composition comprising one or more IGR Deletion Arenaviruses according to claim 1.

13. The composition according to claim 12, and further comprising pharmaceutically acceptable carrier.

14. The composition according to claim 12, and further comprising an adjuvant.

15. A live attenuated vaccine which comprises one or more IGR Deletion Arenaviruses according to claim 1.

16. A method of immunizing a subject against an Arenavirus, which comprises administering to the subject an immunogenic amount of one or more IGR Deletion Arenaviruses according to claim 1 or a composition thereof.

17. A method of treating, inhibiting, or reducing an Arenavirus infection or a disease caused by an Arenavirus in a subject, which comprises administering to the subject an effective amount of one or more IGR Deletion Arenaviruses according to claim 1 or a composition thereof.

18. The method according to claim 16, wherein the administration is before, during, and/or after exposure to the Arenavirus.

19. The method according to claim 16, wherein the Arenavirus and the IGR Deletion Arenavirus are of different viral species.

20. The method according to claim 19, wherein the Arenavirus is a Guanarito virus or a Junin virus and the IGR Deletion Arenavirus is a Machupo virus.

21. The method according to claim 16, wherein the one or more IGR Deletion Arenaviruses elicits a protective immune response against the Arenavirus when administered to the subject.