Lassa fever (LF), a zoonotic disease caused by Lassa virus (LASV), can lead to acute hemorrhagic fever with a case fatality rate (CFR) of up to 50% [1
]. The estimated annual incidence of LF across West African countries including Ghana, Guinea, Mali, Benin, Liberia, Sierra Leone, Togo, and Nigeria has been reported as high as 300,000 infections and 5000–10,000 deaths, but these figures are most likely underestimates due to inadequate diagnosis and surveillance [2
]. Based on prospective studies performed in Guinea, Sierra Leone, Liberia, and Nigeria, it was estimated that 59 million people are at risk of LASV infections, with as many as 67,000 deaths per year [6
]. The 2018 outbreak in Nigeria resulted in 3498 suspected cases in 22 states and 171 deaths with a CFR of 27% based on confirmed cases [7
]. The fact that a virus that was first discovered in 1969 in Lassa village in Nigeria is still capable of causing outbreaks in the same geographical regions indicates challenges in eradicating its animal reservoir.
The main animal reservoir of LASV is the multimammate rat (Mastomys natalensis
), which is common in West Africa, but the potential transmission of LASV to new rodent hosts could have serious implications for its spread beyond West Africa [8
]. LASV is mainly transmitted to humans by consumption of contaminated food, by contact with rodent urine and droppings, or by inhalation of virus particles from the excreta of infected animals; it can also be transmitted from human to human through nosocomial infections [9
]. Most LASV infections are asymptomatic or cause only mild symptoms, but some infections lead to multi-organ failure, fever, and, on occasion, hemorrhage and death [10
LASV is a member of the family Arenaviridae
and genus Mammarenavirus,
which includes Old World and New World arenaviruses [12
]. Furthermore, LASV strains are classified into four lineages, I–IV (with a newly proposed fifth lineage from Cote d’lvoire and Mali) based on their genetic variations [12
]. The LASV genome is composed of two ambisense RNA segments. The large (L) segment of 7.2 kb in length encodes the viral RNA-dependent RNA polymerase protein and the zinc finger (Z) protein, a multifunctional matrix protein [14
]. The small (S) segment of 3.4 kb in length encodes the nucleoprotein (NP) and glycoprotein precursor (GPC). GPC is cleaved post-translationally to form a trimer of hetero-trimers, each consisting of a receptor binding protein (GP1), a fusion protein (GP2) and a stable signal peptide (SSP) [16
]. GPC sequences, shown previously to be required for full protection, have been used as the protective antigen in the construction of a large number of vaccine candidates delivered by various platforms. Some of these candidates have undergone preclinical evaluations in mice, guinea pigs, and non-human primates (NHP) [12
], and a DNA vaccine has entered clinical trials [18
Here, we describe the construction, characterization and efficacy of a Lassa vaccine candidate that generates virus-like particles (VLPs) by expression of Lassa GPC and Z proteins from our Modified Vaccinia Ankara (MVA) vector. It has been previously demonstrated that co-expression of Z and GP proteins leads to the formation of VLPs [19
]. A murine model was chosen initially to establish dose and route of vaccination and whether our constructs could elicit any protective immunity. In this model, ML29, an attenuated Mopeia -Lassa (MOP/LAS) reassortant, is delivered into the brain and primarily infects brain capillary endothelial and glial cells that present LASV antigens, triggering an influx of lymphocytes 5–6 days later. The infiltrating cells contribute to encephalitis that is lethal unless the mice had been previously vaccinated against LASV (Djavani and Salvato, unpublished). The intra-cerebral (IC) delivery we used was not “natural”, since it models brain infections that are rare in LF, but it allows a rapid and simple read-out for whether or not we elicited a protective immunity. Here, we show that a single dose of the vaccine conferred full protection against a lethal challenge dose of the ML29 virus delivered IC.
LF has a greater human impact than any other hemorrhagic fever, with the exception of Dengue fever [6
]. Despite the severe impact of LF on populations in West Africa, no effective and practical medical countermeasure is available [18
]. The one available treatment is the antiviral drug ribavirin, which is effective only if given within six days after infection [18
]. A vaccine against LASV would be an ideal solution to the problem of LF, especially given the growing threat of outbreaks due to globalization and increased travel [27
]. Several groups have developed vaccine candidates that have shown activity against LASV in animal models [20
], but only one has progressed to the clinic [18
]. Although a multi-dose immunization regiment is logistically feasible for medical providers and military personnel, a single dose vaccine is critical for epidemic response and is preferable and much more practical in under-resourced endemic areas. LF has been listed in the WHO R&D Blueprint among the 10 diseases and pathogens to prioritize for research and development in public health emergency contexts [28
]. These diseases are identified as those that pose a public health risk because of their epidemic potential and for which there are insufficient countermeasures. For this reason, CEPI (Coalition for Epidemic Preparedness Innovations), founded to address the threat of epidemic diseases, has set aside a major portion of its $
1 billion budget for the development of vaccines against LF [29
GEO-LM01 provides a unique combination of advantages required for visitors or residents of LASV endemic countries. The potential for GEO-LM01 to protect after a single dose is favorable for travelers and military personnel who require a rapid onset of immunity (ideally ~<2 weeks post vaccination) as well as for epidemic/endemic vaccinations (due to the urgency of epidemic response and the complexity of locating vaccinated subjects in rural villages to administer a second dose for routine vaccination campaigns).
The GEO-LM01 reported here is designed to produce secreted as well as cell-associated LASV proteins in VLP (GPC+Z) formations in vaccinated subjects. This vaccine thereby provides two sources of viral antigens that mimic a natural viral infection promoting predominantly a balanced T cell response, which is critical for protection against LASV infection [30
In our murine model, antibody responses occur too late to protect from an acute viral infection. We assessed binding antibodies in ML29-coated ELISA plates that are able to detect both the GP and Z components of VLP. Unfortunately, half of the Z protein is comprised of a RING structure that is common to many ligases and transcription factors in the normal cell, so it cross-reacts with many other proteins. In a recent publication [30
], we posit that the value of the Z in VLP vectors is mostly to boost immunity by forming a 3-D structure with GP since conformational antigens are more immunogenic than linear antigens (boosting both CD4 and B cell responses).
As a vaccine candidate, GEO-LM01 has a unique combination of advantages that could potentially meet the preferred Target Product Profiles (TPP) of a LASV vaccine set by the WHO for both non-emergency (Preventive Use) and emergency settings (Reactive/Outbreak use) [28
]. These include the immunological advantages of a live but safe (replication-deficient) vector that produces conformationally relevant VLPs; inclusion of multiple genes of LASV (GPC and Z) for induction of potentially broad immunity; the ability to immunize sequentially or concurrently with other MVA-vectored vaccines without generating any vector immunity [32
] (potentially even combining GEO-LM01 with other hemorrhagic fever vaccines in a multivalent formulation); the likelihood of single-dose protection, as demonstrated with MVA-VLP-EBOV and MVA-Zika vaccines candidates [32
] with the durability of the immune response attributed to MVA-vectored vaccines; and a temperature stable, simple, adjuvant-free presentation.
Considering the high prevalence of HIV in some LASV endemic areas of Sub-Saharan Africa (up to 68% in some populations) [34
], an LASV vaccine must be safe and effective in immunocompromised populations [35
]. Safety for the parental MVA vector was shown in more than 120,000 subjects in Europe, including immunocompromised individuals, during the initial development of MVA as a “safer smallpox vaccine” [36
]. Furthermore, MVA has shown no reproductive toxicity in studies in pregnant rabbits or rats [38
], and was used as a vector for malaria, influenza, and tuberculosis vaccines as well for cancer therapies in over 100 clinical trials since 1999 [32
]. Our MVA-VLP HIV vaccine has demonstrated outstanding safety in clinical trials involving 500 humans [40
], including immunocompromised individuals as well as HIV patients [40
In this paper, we demonstrated that a single dose of GEO-LM01 confered full protection against the challenge virus ML29, which was directly inoculated into the brain. ML29 virus itself confers protection by the peripheral route, but its classification as a Risk group 3 (BSL3) agent creates major challenges for manufacturing in the US; in Europe it is considered Risk group 2 (BSL2). Similarly, commercial yellow fever (YF) vaccines (strains 17D and 17DD), approved more than half a century ago, are lethal for immunocompetent mice when inoculated by the IC route but can confer protection when administered by peripheral routes [43
]. These strains are used as challenge viruses for the evaluation of new vaccines against YF virus. Our future work will continue in Hartley guinea pigs and NHP models (e.g., in cynomolgous macaques where signs of LASV infection mirror human disease) with live LASV under high laboratory containment, that is, Animal BSL4 (ABSL4) where the challenge virus is fully lethal by the parenteral route.
T cells are known to be a vital component of immune-mediated protection against LASV infection [44
]. T cells also steer the B cell response and, therefore, a thorough investigation of the T cell response yields insight concerning the mechanisms of both cellular and humoral protection. Despite a strong T cell response, especially with CD4+ populations that are double positive for IFNγ and IL2 (an indication of expanding population), at day 11 after single-dose vaccination there was a low level of IgG antibodies produced at this timepoint. While administration of nAbs in the early stages of LASV infection by passive transfer has shown protection in animal models [45
], the protection afforded by prior vaccination is thought to be strictly dependent on a T cell response [30
]. Similarly, multiple vaccine vector platforms that showed high levels of protection in mice, guinea pigs, and NHP have not induced detectable levels of neutralizing Ab (reviewed in Ref 26).
In the 1980s, Clegg and Lloyd [49
] expressed Lassa NP from a vaccinia vector and succeeded in protecting guinea pigs, but not primates, from lethal LF disease. Since then, researchers at Porton Down have used a safer vector, MVA, to express LASV NP and protect guinea pigs from LF disease progression [50
]. Since they elicited both bAb and a robust cell-mediated immunity to NP in the guinea pig model, it is possible that the MVA-LAS NP could be a standalone vaccine.
GPC is the natural target for nAb responses since it contains epitopes involved in both entry and the endosomal fusion process. GPC, however, is highly glycosylated with 11 N-linked carbohydrate sites on each monomer comprising ~25% of the total mass of the protein, shielding critical residues from Ab access. nAb against GPC have been isolated from the sera of convalescent patients, but these are rare and appear very late in infection [51
]. We have consistently elicited excellent functional antibody responses (e.g., bAb, nAb and antibody-dependent cell-mediated cytotoxicity, ADCC) with MVA vaccines that target other viruses such as HIV, Ebola, and Zika [32
]. A recent publication elicits excellent bAb against an MVA vector expressing LASV NP that has no glycan shield [52
]. The low natural bAb against LASV GPC speaks to the effectiveness of the immuno-evasive properties of the protein, including its glycan shield, explaining failures of nAb-based vaccines or therapies using convalescent sera [51
]. The combination of the 33 glycans shield, leaving only a few regions vulnerable to antibody binding, and the metastability of the native protein further explain why bAb responses are altogether difficult to elicit by vaccination. Here, we saw robust bAb at d36 (Figure 4
d), but that could be attributed to the boost from ML29 virus challenge. Neutralizing antibodies have been isolated from LF survivors as patients were recovering from illness during which the humoral response had time to mature [53
]. Recently, by introducing a number of mutations in GPC, this protein could be “locked” in its prefusion state increasing its stability and enabling co-crystallization with human nAb 37.7 H [16
]. We are currently exploring whether replacement of the locked GPC structure with native GPC or any other modifications thereof could result in a viable vaccine candidate, demasking the GPC from carbohydrate shields and exposing more epitopes for inducing binding and neutralizing antibodies in vaccinated hosts.