A Lassa Virus Live-Attenuated Vaccine Candidate Based on Rearrangement of the Intergenic Region

ABSTRACT Lassa virus (LASV) poses a significant public health problem within the regions of Lassa fever endemicity in Western Africa. LASV infects several hundred thousand individuals yearly, and a considerable number of Lassa fever cases are associated with high morbidity and lethality. No approved LASV vaccine is available, and current therapy is limited to an off-label usage of ribavirin that is only partially effective and associated with significant side effects. The impact of Lassa fever on human health, together with the limited existing countermeasures, highlights the importance of developing effective vaccines against LASV. Here, we present the development and characterization of a recombinant LASV (rLASV) vaccine candidate [rLASV(IGR/S-S)], which is based on the presence of the noncoding intergenic region (IGR) of the small (S) genome segment (S-IGR) in both large (L) and S LASV segments. In cultured cells, rLASV(IGR/S-S) was modestly less fit than wild-type rLASV (rLASV-WT). rLASV(IGR/S-S) was highly attenuated in guinea pigs, and a single subcutaneous low dose of the virus completely protected against otherwise lethal infection with LASV-WT. Moreover, rLASV(IGR/S-S) was genetically stable during serial passages in cultured cells. These findings indicate that rLASV(IGR/S-S) can be developed into a LASV live-attenuated vaccine (LAV) that has the same antigenic composition as LASV-WT and a well-defined mechanism of attenuation that overcomes concerns about increased virulence that could be caused by genetic changes in the LAV during multiple rounds of multiplication. IMPORTANCE Lassa virus (LASV), the causative agent of Lassa fever, infects several hundred thousand people in Western Africa, resulting in many lethal Lassa fever cases. No U.S. Food and Drug Administration-licensed countermeasures are available to prevent or treat LASV infection. We describe the generation of a novel LASV live-attenuated vaccine candidate rLASV(IGR/S-S), which is based on the replacement of the large genomic segment noncoding intergenic region (IGR) with that of the small genome segment. rLASV(IGR/S-S) is less fit in cell culture than wild-type virus and does not cause clinical signs in inoculated guinea pigs. Importantly, rLASV(IGR/S-S) protects immunized guinea pigs against an otherwise lethal exposure to LASV.

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A Lassa Fever Live-Attenuated Vaccine Based on Codon Deoptimization of the Viral Glycoprotein Gene

mBio ◽

10.1128/mbio.00039-20 ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 9

Author(s):

Yingyun Cai ◽

Chengjin Ye ◽

Benson Cheng ◽

Aitor Nogales ◽

Masaharu Iwasaki ◽

...

Keyword(s):

Guinea Pigs ◽

Vaccine Candidate ◽

Lassa Fever ◽

Lassa Virus ◽

Off Label Use ◽

Wild Type ◽

Glycoprotein Precursor ◽

Live Attenuated Vaccine ◽

Off Label ◽

Western Africa

ABSTRACT Lassa virus (LASV) is endemic in Western Africa and is estimated to infect hundreds of thousands of individuals annually. A considerable number of these infections result in Lassa fever (LF), which is associated with significant morbidity and a case-fatality rate as high as 69% among hospitalized confirmed patients. U.S. Food and Drug Administration-approved LF vaccines are not available. Current antiviral treatment is limited to off-label use of a nucleoside analogue, ribavirin, that is only partially effective and associated with significant side effects. We generated and characterized a recombinant LASV expressing a codon-deoptimized (CD) glycoprotein precursor gene (GPC), rLASV-GPC/CD. Comparison of growth kinetics and peak titers showed that rLASV-GPC/CD is slightly attenuated in cell culture compared to wild-type (WT) recombinant LASV (rLASV-WT). However, rLASV-GPC/CD is highly attenuated in strain 13 and Hartley guinea pigs, as reflected by the absence of detectable clinical signs in animals inoculated with rLASV-GPC/CD. Importantly, a single subcutaneous dose of rLASV-GPC/CD provides complete protection against an otherwise lethal exposure to LASV. Our results demonstrate the feasibility of implementing a CD approach for developing a safe and effective LASV live-attenuated vaccine candidate. Moreover, rLASV-GPC/CD might provide investigators with a tool to safely study LASV outside maximum (biosafety level 4) containment, which could accelerate the elucidation of basic aspects of the molecular and cell biology of LASV and the development of novel LASV medical countermeasures. IMPORTANCE Lassa virus (LASV) infects several hundred thousand people in Western Africa, resulting in many lethal Lassa fever (LF) cases. Licensed LF vaccines are not available, and anti-LF therapy is limited to off-label use of the nucleoside analog ribavirin with uncertain efficacy. We describe the generation of a novel live-attenuated LASV vaccine candidate. This vaccine candidate is based on mutating wild-type (WT) LASV in a key region of the viral genome, the glycoprotein precursor (GPC) gene. These mutations do not change the encoded GPC but interfere with its production in host cells. This mutated LASV (rLASV-GPC/CD) behaves like WT LASV (rLASV-WT) in cell culture, but in contrast to rLASV-WT, does not cause disease in inoculated guinea pigs. Guinea pigs immunized with rLASV-GPC/CD were protected against an otherwise lethal exposure to WT LASV. Our results support the testing of this candidate vaccine in nonhuman primate models ofLF.

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A Live Attenuated Vaccine for Lassa Fever Made by Reassortment of Lassa and Mopeia Viruses

Journal of Virology ◽

10.1128/jvi.79.22.13934-13942.2005 ◽

2005 ◽

Vol 79 (22) ◽

pp. 13934-13942 ◽

Cited By ~ 95

Author(s):

Igor S. Lukashevich ◽

Jean Patterson ◽

Ricardo Carrion ◽

Dmitry Moshkoff ◽

Anysha Ticer ◽

...

Keyword(s):

Guinea Pigs ◽

Rhesus Macaques ◽

Detailed Examination ◽

Lassa Fever ◽

Lymphocytic Choriomeningitis ◽

Cell Mediated Immunity ◽

Lassa Virus ◽

Live Attenuated Vaccine ◽

Attenuated Vaccine ◽

Primary Virus

ABSTRACT Lassa virus (LASV) and Mopeia virus (MOPV) are closely related Old World arenaviruses that can exchange genomic segments (reassort) during coinfection. Clone ML29, selected from a library of MOPV/LASV (MOP/LAS) reassortants, encodes the major antigens (nucleocapsid and glycoprotein) of LASV and the RNA polymerase and zinc-binding protein of MOPV. Replication of ML29 was attenuated in guinea pigs and nonhuman primates. In murine adoptive-transfer experiments, as little as 150 PFU of ML29 induced protective cell-mediated immunity. All strain 13 guinea pigs vaccinated with clone ML29 survived at least 70 days after LASV challenge without either disease signs or histological lesions. Rhesus macaques inoculated with clone ML29 developed primary virus-specific T cells capable of secreting gamma interferon in response to homologous MOP/LAS and heterologous MOPV and lymphocytic choriomeningitis virus. Detailed examination of two rhesus macaques infected with this MOPV/LAS reassortant revealed no histological lesions or disease signs. Thus, ML29 is a promising attenuated vaccine candidate for Lassa fever.

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Vaccines inducing immunity to Lassa virus glycoprotein and nucleoprotein protect macaques after a single shot

Science Translational Medicine ◽

10.1126/scitranslmed.aaw3163 ◽

2019 ◽

Vol 11 (512) ◽

pp. eaaw3163 ◽

Cited By ~ 12

Author(s):

Mathieu Mateo ◽

Stéphanie Reynard ◽

Xavier Carnec ◽

Alexandra Journeaux ◽

Nicolas Baillet ◽

...

Keyword(s):

T Cell ◽

Immune Responses ◽

Measles Virus ◽

Vaccine Candidate ◽

Lassa Fever ◽

Lassa Virus ◽

Single Shot ◽

Efficacy Analysis ◽

Western Africa ◽

Efficacious Vaccine

Lassa fever is a major threat in Western Africa. The large number of people living at risk for this disease calls for the development of a vaccine against Lassa virus (LASV). We generated live-attenuated LASV vaccines based on measles virus and Mopeia virus platforms and expressing different LASV antigens, with the aim to develop a vaccine able to protect after a single shot. We compared the efficacy of these vaccines against LASV in cynomolgus monkeys. The vaccines were well tolerated and protected the animals from LASV infection and disease after a single immunization but with varying efficacy. Analysis of the immune responses showed that complete protection was associated with robust secondary T cell and antibody responses against LASV. Transcriptomic and proteomic analyses showed an early activation of innate immunity and T cell priming after immunization with the most effective vaccines, with changes detectable as early as 2 days after immunization. The most efficacious vaccine candidate, a measles vector simultaneously expressing LASV glycoprotein and nucleoprotein, has been selected for further clinical evaluation.

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Enhancing the Thermostability and Immunogenicity of a Respiratory Syncytial Virus (RSV) Live-Attenuated Vaccine by Incorporating Unique RSV Line19F Protein Residues

Journal of Virology ◽

10.1128/jvi.01568-17 ◽

2017 ◽

Vol 92 (6) ◽

Cited By ~ 7

Author(s):

Christina A. Rostad ◽

Christopher C. Stobart ◽

Sean O. Todd ◽

Samuel A. Molina ◽

Sujin Lee ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Vaccine Candidate ◽

Nonstructural Protein ◽

Wild Type ◽

Live Attenuated Vaccine ◽

F Protein ◽

Attenuated Vaccine ◽

Cotton Rats ◽

Syncytial Virus ◽

Tract Infections

ABSTRACTRespiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in infants, and an effective vaccine is not yet available. We previously generated an RSV live-attenuated vaccine (LAV) candidate, DB1, which was attenuated by a low-fusion subgroup B F protein (BAF) and codon-deoptimized nonstructural protein genes. DB1 was immunogenic and protective in cotton rats but lacked thermostability and stability of the prefusion conformation of F compared to strains with the line19F gene. We hypothesized that substitution of unique residues from the thermostable A2-line19F strain could thermostabilize DB1 and boost its immunogenicity. We therefore substituted 4 unique line19F residues into the BAF protein of DB1 by site-directed mutagenesis and rescued the recombinant virus, DB1-QUAD. Compared to DB1, DB1-QUAD had improved thermostability at 4°C and higher levels of prefusion F as measured by enzyme-linked immunosorbent assays (ELISAs). DB1-QUAD was attenuated in normal human bronchial epithelial cells, in BALB/c mice, and in cotton rats but grew to wild-type titers in Vero cells. In mice, DB1-QUAD was highly immunogenic and generated significantly higher neutralizing antibody titers to a panel of RSV A and B strains than did DB1. DB1-QUAD was also efficacious against wild-type RSV challenge in mice and cotton rats. Thus, substitution of unique line19F residues into RSV LAV DB1 enhanced vaccine thermostability, incorporation of prefusion F, and immunogenicity and generated a promising vaccine candidate that merits further investigation.IMPORTANCEWe boosted the thermostability and immunogenicity of an RSV live-attenuated vaccine candidate by substituting 4 unique residues from the RSV line19F protein into the F protein of the heterologous vaccine strain DB1. The resultant vaccine candidate, DB1-QUAD, was thermostable, attenuatedin vivo, highly immunogenic, and protective against RSV challenge in mice and cotton rats.

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Immunogenicity, Genetic Stability, and Protective Efficacy of a Recombinant, Chimeric Yellow Fever-Japanese Encephalitis Virus (ChimeriVax-JE) as a Live, Attenuated Vaccine Candidate against Japanese Encephalitis

Virology ◽

10.1006/viro.1999.9695 ◽

1999 ◽

Vol 257 (2) ◽

pp. 363-372 ◽

Cited By ~ 123

Author(s):

F. Guirakhoo ◽

Z.-X. Zhang ◽

T.J. Chambers ◽

S. Delagrave ◽

J. Arroyo ◽

...

Keyword(s):

Japanese Encephalitis Virus ◽

Yellow Fever ◽

Japanese Encephalitis ◽

Genetic Stability ◽

Vaccine Candidate ◽

Protective Efficacy ◽

Encephalitis Virus ◽

Live Attenuated Vaccine ◽

Attenuated Vaccine

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Attenuated Replication of Lassa Virus Vaccine Candidate ML29 in STAT-1-/- Mice

Pathogens ◽

10.3390/pathogens8010009 ◽

2019 ◽

Vol 8 (1) ◽

pp. 9 ◽

Cited By ~ 5

Author(s):

Dylan Johnson ◽

Jenny Jokinen ◽

Igor Lukashevich

Keyword(s):

Vaccine Development ◽

Vaccine Candidate ◽

Small Animal ◽

Vero Cells ◽

Lassa Fever ◽

Effective Vaccine ◽

Cell Mediated Immunity ◽

Lassa Virus ◽

Cell Responses ◽

Fold Reduction

Lassa virus (LASV), a highly prevalent mammalian arenavirus endemic in West Africa, can cause Lassa fever (LF), which is responsible for thousands of deaths annually. LASV is transmitted to humans from naturally infected rodents. At present, there is not an effective vaccine nor treatment. The genetic diversity of LASV is the greatest challenge for vaccine development. The reassortant ML29 carrying the L segment from the nonpathogenic Mopeia virus (MOPV) and the S segment from LASV is a vaccine candidate under current development. ML29 demonstrated complete protection in validated animal models against a Nigerian strain from clade II, which was responsible for the worst outbreak on record in 2018. This study demonstrated that ML29 was more attenuated than MOPV in STAT1-/- mice, a small animal model of human LF and its sequelae. ML29 infection of these mice resulted in more than a thousand-fold reduction in viremia and viral load in tissues and strong LASV-specific adaptive T cell responses compared to MOPV-infected mice. Persistent infection of Vero cells with ML29 resulted in generation of interfering particles (IPs), which strongly interfered with the replication of LASV, MOPV and LCMV, the prototype of the Arenaviridae. ML29 IPs induced potent cell-mediated immunity and were fully attenuated in STAT1-/- mice. Formulation of ML29 with IPs will improve the breadth of the host’s immune responses and further contribute to development of a pan-LASV vaccine with full coverage meeting the WHO requirements.

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Recombinant Lassa Virus Expressing Green Fluorescent Protein as a Tool for High-Throughput Drug Screens and Neutralizing Antibody Assays

Viruses ◽

10.3390/v10110655 ◽

2018 ◽

Vol 10 (11) ◽

pp. 655 ◽

Cited By ~ 6

Author(s):

Yíngyún Caì ◽

Masaharu Iwasaki ◽

Brett Beitzel ◽

Shuīqìng Yú ◽

Elena Postnikova ◽

...

Keyword(s):

Green Fluorescent Protein ◽

High Throughput ◽

Reverse Genetics ◽

Fluorescent Protein ◽

Cultured Cells ◽

Neutralizing Antibody ◽

Quantitative Detection ◽

Lassa Virus ◽

Wild Type ◽

Green Fluorescent

Lassa virus (LASV), a mammarenavirus, infects an estimated 100,000–300,000 individuals yearly in western Africa and frequently causes lethal disease. Currently, no LASV-specific antivirals or vaccines are commercially available for prevention or treatment of Lassa fever, the disease caused by LASV. The development of medical countermeasure screening platforms is a crucial step to yield licensable products. Using reverse genetics, we generated a recombinant wild-type LASV (rLASV-WT) and a modified version thereof encoding a cleavable green fluorescent protein (GFP) as a reporter for rapid and quantitative detection of infection (rLASV-GFP). Both rLASV-WT and wild-type LASV exhibited similar growth kinetics in cultured cells, whereas growth of rLASV-GFP was slightly impaired. GFP reporter expression by rLASV-GFP remained stable over several serial passages in Vero cells. Using two well-characterized broad-spectrum antivirals known to inhibit LASV infection, favipiravir and ribavirin, we demonstrate that rLASV-GFP is a suitable screening tool for the identification of LASV infection inhibitors. Building on these findings, we established a rLASV-GFP-based high-throughput drug discovery screen and an rLASV-GFP-based antibody neutralization assay. Both platforms, now available as a standard tool at the IRF-Frederick (an international resource), will accelerate anti-LASV medical countermeasure discovery and reduce costs of antiviral screens in maximum containment laboratories.

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A recombinant vesicular stomatitis-based Lassa fever vaccine elicits rapid and long-term protection from lethal Lassa virus infection in guinea pigs

npj Vaccines ◽

10.1038/s41541-019-0104-x ◽

2019 ◽

Vol 4 (1) ◽

Cited By ~ 10

Author(s):

Derek R. Stein ◽

Bryce M. Warner ◽

Geoff Soule ◽

Kevin Tierney ◽

Kathy L. Frost ◽

...

Keyword(s):

Virus Infection ◽

Guinea Pigs ◽

Lassa Fever ◽

Lassa Virus ◽

Vesicular Stomatitis

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Pichinde Virus Infection of Outbred Hartley Guinea Pigs as a Surrogate Animal Model for Human Lassa Fever: Histopathological and Immunohistochemical Analyses

Pathogens ◽

10.3390/pathogens9070579 ◽

2020 ◽

Vol 9 (7) ◽

pp. 579 ◽

Cited By ~ 1

Author(s):

Wun-Ju Shieh ◽

Shuiyun Lan ◽

Sherif R. Zaki ◽

Hinh Ly ◽

Yuying Liang

Keyword(s):

Animal Model ◽

Surrogate Model ◽

Guinea Pigs ◽

Virulent Strain ◽

Small Animal ◽

Immunohistochemical Analysis ◽

Lassa Fever ◽

Lassa Virus ◽

Pichinde Virus ◽

Guinea Pig Model

Lassa virus (LASV) is a mammarenavirus (arenavirus) that causes zoonotic infection in humans that can lead to fatal hemorrhagic Lassa fever (LF) disease. Currently, there are no FDA-approved vaccines or therapeutics against LASV. Development of treatments against LF and other related arenavirus-induced hemorrhagic fevers (AHFs) requires relevant animal models that can recapitulate clinical and pathological features of AHF diseases in humans. Laboratory mice are generally resistant to LASV infection, and non-human primates, while being a good animal model for LF, are limited by their high cost. Here, we describe a small, affordable, and convenient animal model that is based on outbred Hartley guinea pigs infected with Pichinde virus (PICV), a mammarenavirus that is non-pathogenic in humans, for use as a surrogate model of human LF. We conducted a detailed analysis of tissue histopathology and immunohistochemical analysis of different organs of outbred Hartley guinea pigs infected with different PICV strains that show differential disease phenotypes and pathologies. Comparing to infection with the avirulent PICV strain (P2 or rP2), animals infected with the virulent strain (P18 or rP18) show extensive pathological changes in different organs that sustain high levels of virus replication. The similarity of tissue pathology and viral antigen distribution between the virulent PICV–guinea pig model and lethal human LASV infection supports a role of this small animal model as a surrogate model of studying human LF in order to understand its pathogenesis and for evaluating potential preventative and therapeutic options against AHFs.

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