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. Author manuscript; available in PMC: 2021 Mar 23.
Published in final edited form as: Vaccine. 2020 Feb 25;38(14):2949–2959. doi: 10.1016/j.vaccine.2020.02.053

Bivalent Junin & Machupo experimental vaccine based on alphavirus RNA replicon vector

Dylan M Johnson a, Jenny D Jokinen b, Min Wang b, Tia Pfeffer b, Irina Tretyakova c, Ricardo Carrion Jr d, Anthony Griffiths d,*, Peter Pushko c, Igor S Lukashevich b,**
PMCID: PMC7112472  NIHMSID: NIHMS1567427  PMID: 32111526

Abstract

Junin (JUNV) and Machupo (MACV), two mammalian arenaviruses placed on the 2018 WHO watch list, are prevalent in South America causing Argentine and Bolivian hemorrhagic fevers (AHF and BHF), respectively. The live attenuated JUNV vaccine, Candid #1, significantly reduced the incidence of AHF. Vaccination induces neutralizing antibody (nAb) responses which effectively target GP1 (the viral attachment glycoprotein) pocket which accepts the tyrosine residue of the cellular receptor, human transferrin receptor 1 (TfR1). In spite of close genetic relationships between JUNV and MACV, variability in the GP1 receptor binding site (e.g., MACV GP1 loop 10) results in poor MACV neutralization by Candid #1-induced nAbs. Candid #1 is not recommended for vaccination of children younger than 15 years old (a growing “at risk” group), pregnant women, or other immunocompromised individuals. Candid #1’s primary reliance on limited missense mutations for attenuation, genetic heterogeneity, and potential stability concerns complicate approval of this vaccine in the US. To address these issues, we applied alphavirus RNA replicon vector technology based on the human Venezuelan equine encephalitis vaccine (VEEV) TC-83 to generate replication restricted virus-like-particles vectors (VLPVs) simultaneously expressing cellular glycoprotein precursors (GPC) of both viruses, JUNV and MACV. Resulting JV&MV VLPVs were found safe and immunogenic in guinea pigs. Immunization with VLPVs induced humoral responses which correlated with complete protection against lethal disease after challenge with pathogenic strains of JUNV (Romero) and MACV (Carvallo).

Keywords: South American Hemorrhagic Fevers, Junin and Machupo arenaviruses, Bivalent vaccine, Alphavirus RNA replicons

1. Introduction

The genus Mammarenavirus of the Arenaviridae family accommodates two groups of rodent-borne viruses: the New World (NW) arenaviruses circulated in Americas; and the Old World (OW) arenaviruses found in Africa and Asia [1]. Mammalian arenaviruses are enveloped RNA viruses with two-segmented ambisense genomes. The large (L) RNA encodes for L protein (RNA-dependent RNA polymerase) and for Z (matrix) protein. The small (S) RNA encodes for nucleoprotein (NP) and glycoprotein precursor (GPC), which is post-translationally processed into stable signal peptide (SSP) and the mature GP1 (attached protein) and GP2 (fusion protein).

Pathogenic arenaviruses can cause human infections with clinical manifestations vary from flu-like illness and meningo-encephalitis (caused by LCMV, lymphocytic choriomeningitis virus, prototypic arenavirus) to severe hemorrhagic fevers (HFs) posing significant threats to public health and national security. Lassa Fever (LF) is the most prevalent OW viral HF in West Africa. Argentine and Bolivian HFs (AHF and BHF, respectively) are the most prevalent NW arenavirus infections in South America [2]. LASV, Junin (JUNV), and Machupo (MACV), causative agents of LF, AHF, and BHF, respectively, are recognized as category A priority pathogens [2]. The World Health Organization (WHO) included LF in the Blueprint List of Priority Diseases and placed HFs caused by other pathogenic mammalian arenaviruses including JUNV and MACV on “watch list” [3].

JUNV and MACV are genetically closely related and induce clinically similar diseases [2]. Both viruses use human transferrin receptor 1 (TfR1) for host cell entry [4], share 69% amino acid sequence identity within the GPC, and have similar GP1 crystal structure [5–7]. The first cases of AHF were reported in the 1950s in rural areas near Buenos Aires. The AHF endemic area was extended further northwest and was associated with the movement of the natural host, Calomys musculinus. The disease occurred seasonally and involved a variable number of cases from a historically high of 3,500 to few dozen after introduction of Candid #1 vaccination [8, 9].

BHF was recognized in 1959, and the etiological agent, MACV, was isolated in 1963 [10, 11]. At the beginning of the 1970s, outbreaks involving around 1,000 patients occurred in the Beni district of Bolivia. During the 1970–90s, sporadic cases and small outbreaks occurred in different communities of the Beni district. The natural host of BHF, C. callosus, occupied wide geographical areas including parts of Argentina, Bolivia, Brazil, and Paraguay. Since 2006, BHF has re-emerged with 200 cases in 2008 [2, 12–14].

Candid #1, the first live-attenuated arenaviral vaccine, was licensed in Argentina in 2006. Candid #1 vaccination resulted in significant reduction of incidence of AHF [15, 16]. The vaccine was developed by a “classical” attenuation approach using serial passages of virulent JUNV. These passages resulted in 17 amino acid substitutions distinguishing Candid #1 from parental JUNV XJ13 [17, 18]. The F427I substitution in transmembrane domain of GP2 has been identified as the major factor responsible for neurovirulence attenuation in mice [19]. A single mutation in the transmembrane domain of MACV GP2 also resulted in attenuation in mice [20]. In guinea pigs, JUNV GPC drives attenuation of Candid #1 as well [21]. However, the F421I mutation alone was insufficient to prevent mild disease in this model. Additional mutations seems to contribute to Candid #1 attenuation in guinea pigs [22].

The JUNV-specific neutralizing antibody (nAb) against GPC is the major factor responsible for elimination of virus [8, 23, 24]. A single intramuscular injection of Candid #1 induced JUNV-specific nAb in 91.1% of vaccinees resulting in 95% vaccine efficacy [25]. Treatment of patients with immune plasma was highly effective as well, however, resulted in a late neurological syndrome observed in 10% of treated patients [26]. Anti- JUNV GPC mAbs were also highly effective in post-exposure treatment [27].

The crystal structure analysis revealed that Abs from AHF survivors targeted the TfR1 binding site on the GP1 and blocked viral entry [6, 28, 29]. The receptor binding site (RBS) accessibility for Abs explained high therapeutic efficiency of immune plasma and of Candid #1 efficacy. Likewise, experimental NHPs infected with MACV and treated with immune IgG from BHF patients were also protected against fatal BHF [30]. Similarly, in guinea pigs, nAbs against MACV GPC were involved in virus control [31–33].

Despite the structural similarity within GP1, cross-neutralization between anti-GPC Abs targeting JUNV and MACV is limited. JUNV GPC Abs were unable to neutralize MACV [33–35]. Structural analysis documented that a loop 10 (L10) protruding from the GP1 core to cellular TfR1 receptor is uniquely presented in MACV GP1 and restricted RBS accessibility for JUNV GPCspecific Abs [29, 34]. As expected, most mAbs generated against MACV GPC were highly specific and bind only to homologous GPC [36].

Candid #1 is not recommended for the vaccination of children less than 15 years of age, pregnant women and immunocompromised individuals. The primary reliance on limited missense mutations for Candid #1 attenuation, genetic heterogeneity, and lack of FDA-compliant documentation to track the passage history of Candid #1 complicate approval of this vaccine in the US [8, 37]. Alphavirus RNA replicon technology provides a reasonable compromise, in terms of safety and immunogenicity, between live-attenuated and inactivated vaccines. Encapsulated alphavirus replicons expressing foreign gene(s) of interests are single-cycle virus-like-particles vehicles, vectors (VLPVs). They are not able to spread beyond the initially infected cells, but can efficiently deliver and transduce the gene(s) of interest into target cells (e.g., dendritic cells, DCs). Numerous alphavirus replicon-based vaccine candidates are in pre-clinical and clinical development [38]. We have used an advanced alphavirus RNA replicon technology based on the human Venezuelan equine encephalitis vaccine (VEEV) TC-83 [39] to generate bi-cistronic RNA replicons expressing multiple GPCs derived from pathogenic arenaviruses [40, 41]. Here, we present results of proof-of-concept immunogenicity and efficacy studies of experimental bivalent JUNV&MACV vaccine in the guinea pig model of AHF and BHF.

2. Materials and methods

2.1. Cell lines and viruses

Vero E6 and BSC-40 cells (from ATCC) were maintained DMEM/F-12 medium with 10% fetal calf serum (FCS), 1X antibiotic-antimycotic, and 1X Glutamax (ThermoFisher). CHO-K1 cells (ATCC) were grown in F12K Medium (ATCC 30–2004) with 10% FCS. JUNV Candid #1 was received from Dr. Paessler (UTMB, Galveston, TX). Pathogenic JUNV (Romero) and MACV (Carvallo) were from Centers for Disease Control and Prevention (CDC, Atlanta, GA). MACV/Carvallo was passaged twice in Vero cells to prepare challenge stock at Texas Biomedical Research Institute (TBRI, San Antonio, TX). Titration of JUNV and MACV was performed by infecting Vero E6 monolayers under agarose overlay [42]. Inactivated (gamma-irradiated) MACV (Carvallo) and recombinant vaccinia virus encoding MACV GPC (rVACV-MACV GPC) [43] were received from BEI Resources.

2.2. Generation of alphavirus replicon vector simultaneously expressing JUNV and MACV GPCs (JV&MV VLPVs)

JUNV (AY746353) and MACV (AY619643.1) GPC genes were synthesized and human-codon optimized by GeneArt AG (www.geneart.com) using a proprietary algorithm. Cis-acting sequences (e.g., splice sites, poly A signals, TATA boxes) negatively impacting gene expression were also removed. The GC-content was adjusted to prolong mRNA half-life. Resulting sequences (GeneArt # 0712227 and 0712226, for JUNV and MACV, respectively) had improved codon adaptation index (CAI) values of 0.98. Codon-optimized genes were cloned in the VEEV TC-83 backbone to generate bi-cistronic replicons expressing JUNV and MACV GPCs from individual TC-83 sub-genomic 26S promoters [40, 41]. For packaging into VLPVs, alphavirus capsid and glycoproteins were supplied in trans from two helper RNAs (Fig. 1A). Optimized RNA formulations containing JV&MV GPC vector and helpers were transfected into CHO-K1 cells. Encapsulated replicons were purified from culture medium and formulated in PBS. Titration of JV&MV VLPVs was performed in immunofluorescence assay (IFA). VLPV stocks with titers 1.0 × 107 - 1.0 × 108 fluorescent focus unit (FFU) per ml were stored at – 80°C before use.

Fig. 1.

Fig. 1.

Fig. 1.

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Design of bi-cistronic virus-like particles vectors (VLPVs) and co-expression of JUNV and MACV GPCs in JUNV- and VLPV-infected cells. Panel (A), bi-vistronic RNA replicons and packaging helpers derived from the human VEEV TC-83 IND vaccine. Codon optimized JUNV and MACV GPC genes were cloned into TC-83 vector downstream from the TC-83 sub-genomic 26S promoters. Bi-cistronic vector and packaging helpers were co-transfected into CHO-K1 cells and encapsulated replicons expressing both genes, JUNV GPC and MACV GPC (JV&MV VLPVs), were purified from culture medium. Vero cells were infected with JUNV (Candid #1), panel (B), or with JV&MV VLPVs, panels (C) and (D). On day 2 after infection, cells were stained with anti-JUNV GPC mAb, clone LD05-BF09, (Fig. 1B, top panels) or with affinity-purified polyclonal Ab against MACV GP1-derived peptide 214Y-C229 (Fig. 1B, low panels) and with DAPI. Fig. 1C, expression of JUNV GPC (left panel), MACV GPC (middle panel), and co-staining of both GPCs (right panel.) Co-expression of JUNV and MACV GPCs (in yellow) in VLPV-exposed Vero cells are arrowed. Panels (D), infected and mock-infected cells were stained with JUNV clone LD05-BF09 (Di) or with rabbit anti-MACV GP-1 polyclonal Ab (Dii) and subsequently stained with Alexa Flour anti-mouse and Texas Red anti-Rabbit Ab and analyzed on flow cytometer. 104 cells were counted per sample. Gray and dark lines are untreated and isotope control staining, respectively.

2.3. Production of MACV GP1-specific affinity-purified polyclonal antibody, IFA, flow cytometry

A 16-mer peptide 214YLTINQCGDPSSFDYC229 (214Y-C229) derived from MACV GP1 loop 10 [28] was synthesized (GenScript, Piscataway, NJ, USA), conjugated with Keywhole limpet hemocyanin (KLH), formulated in incomplete Freund’s adjuvant, and used for sub-cutaneous (SC) immunization of two rabbits. After the 3rd immunization, both rabbits had titers ≥ 1:512,000 in IgG ELISA. Affinity-purified anti-MACV-GP1 Ab (≥ 2 mg/rabbit) demonstrated strong binding to MACV GPC in Western blot (not shown), did not cross react with JUNV GPC (Fig. 1B), and specifically interacted with MACV GPC in VACV-MACV GPC- and JV&MV VLPV-infected cells (Fig. 1C). Murine mAb specific for JUNV GPC (LD05-BF09) and JUNV NP (IC06-BE10) were purchased from BEI Resources. Identity of bivalent JV&MV VLPVs was assessed by IFA as previously described [41]. Briefly, Vero E6 cells in Lab-TE II chamber slides were infected with JV&MV VLPVs at a multiplicity of infection (MOI) of 1 and incubated at 37° C. At D2, cells were fixed with 4% paraformaldehyde, permeabilized, and stained with primary Ab (anti-JUNV GPC mAb and rabbit anti-MACV GP1, 1:100 dilution) and with secondary Abs (Alexa Fluor 488 anti-mouse IgG2a, A21131 from Molecular Probes; Texas Red anti-Rabbit IgG, sc-2780 from Santa Cruz Biotechnology, dilution 1:500). Mounting slides with DAPI were analyzed with a Zeiss AX10 microscope. Expression of virus-specific GPC in infected Vero E6 cells was also measured by flow cytometry (FACScalibur, BD Bioscience, San Diego, CA). JV&MV VLPV-infected cells were incubated with anti-JUNV GPC mAb and with rabbit anti-MACV GP1 affinity purified polyclonal IgG (1:100 dilution). After primary staining, cells were then incubated with secondary anti-mouse Alexa Fluor and anti-rabbit Texas Red (1:500 dilution) and processed for flow cytometry analyzing 10,000 cells per sample by live gating.

2.4. Immunogenicity studies in guinea pigs, assessment of immune responses in IFA, IgG ELISA, and PRNT

All animal protocols were in compliance with the U.S. Department of Agriculture’s (USDA) Animal Welfare Act and the National Institutes of Health, Office of Laboratory Animal Welfare. Hartley guinea pigs (females, 6–10-week-old) were purchased from Charles River (Wilmington, MA) and randomly placed into 4 groups (n=4–6). Group 1 was SC inoculated with JV&MV VLVPs (1 × 107 FFU in 0.1 ml of PBS) and boosted on D28 with the same dose (Fig. 2A). Group 2 received Candid #1 (1 × 103 PFU). Groups 3 and 4 received placebo vaccine (challenge control). Animals were monitored daily and blood samples were collected weekly from the femoral vein. Immunogenicity of JV&MV VLPVs was assessed in IFA, IgG ELISA, and plaque reduction neutralization assay (PRNT). For IFA, BSC-1 cells were infected with rVACV-MACV GPC at a MOI of 10 and incubated for 24 hours. The cells were fixed with 4% paraformaldehyde, blocked with 1% BSA, and incubated with plasma samples (1:40 dilution) from VLPV-vaccinated guinea pigs. Texas Red anti-Rabbit IgG conjugate was used for the 2nd Abs staining (1:400 dilution). Mounting slides with DAPI were covered with glass and analyzed with a Zeiss AX10 microscope. The IgG ELISA was performed as previously described [44]. Inactivated MACV antigen (1:1000 dilution) was used to cover wells of micro-titration plates (overnight at 4° C). Wells were washed with 0.05% Tween-20 in PBS, blocked with 10% nonfat dry milk in PBS/Tween-20 buffer, and 0.1 ml of plasma sample dilutions from vaccinated guinea pigs were added to the 96-well plates. Plates were incubated at 37° C for 2 hours, washed, and anti-guinea pig IgG peroxidase conjugate was added (1:5000 dilution). The substrate for the peroxidase enzyme (TMB) was added and color development was measured in a plate reader at 450 nm. NAbs were measured by traditional PRNT with a constant dose of the virus and serial 1-log dilutions of plasma from vaccinated guinea pigs [45]. Incubation of virus with Ab dilutions was performed at 37° C for 1 hour. End points were calculated from the highest serum dilution inducing 50% plaque reduction. PCR-based neutralization (PCR-NA) was used for enhanced sensitivity compared to PRNT assay [46, 47]. In brief, Vero E6 cells seeded in 96-well plates were infected with Candid #1 (MOI = 0.1), mixed with 2-fold dilutions of immune sera, and incubated for 1 hour at 37°C. At 48 hours after infection, RNA from infected cells was extracted and subjected to qRT-PCR analysis targeting the JUNV NP gene. The NP RNA copies were normalized to RNA samples from infected cells incubated with appropriate dilutions of nonimmune sera. Serial dilutions of standard JUNV NP cDNA plasmid with known quantities of copies were used to generate standard curve (R2 = 0.96) for quantitation. Three experimental replicates were used for each time-point after immunization to measure nAbs in qPCR-NA. The highest dilution resulted in 50% reduction of NP RNA copy numbers was defined as the neutralization titer in this assay.

Fig. 2.

Fig. 2.

Fig. 2.

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Humoral responses in guinea pigs vaccinated with JV&MV VLPVs. Hartley guinea pigs (n=3–6 animals per group) were vaccinated with experimental JV&MV VLPV vaccine using prime-boost protocol. Panel (A), vaccination-challenge protocol. Guinea pigs were vaccinated with VLPVs (1 ×107 FFU in 0.1 ml of PBS, SC) and boosted with the same dose at week 4. At the same time, control vaccine groups received either a single SC injection of Candid #1 (1×103 PFU in 0.1 ml of PBS, vaccine control) or PBS (mock-vaccine, challenge control group). Animals were weekly bled (on W0, W2-W7, for VLPV-vaccinated animals; and on D7, D14, D21, for Candid 1- and mock-vaccinated guinea pigs) and plasma samples were used for titration of specific Abs. Vaccinated guinea pigs were shipped to ABSL4 facility. After acclimatization, vaccinated animals were challenged with JUNV or MACV as described in Materials and Methods. Panel (B), anti-JUNV nAbs detected in guinea pigs immunized with JV&MV VLPVs in PRNT50. In control guinea pigs vaccinated with Candid 1, PRNT50 titers were 1/160 and 1/320 on D14 and D21 after immunization, respectively. Panel C, anti-JUNV nAbs detected in PCR-based neutralization assay (PCR-NA), in VLPV- (Ci) or in Candid #1-vaccinated (Cii) guinea pigs. Panels (D), IFA staining using plasma samples (W0, W6, W7; dilutions 1:40) from VLPV-immunized animals and cells infected with recombinant vaccinia virus expressing MACV GPC (rVACV-MACV GPC), panels (Di), or cells infected with Candid #1 (Dii). Panel (E), IgG ELISA, using MACV antigen and plasma samples from VLVP- or Candid # 1-immunized guinea pigs. Gamma-irradiated MACV antigen was prepared from infected Vero E6 cell pellets and purified from cell debris (BEI Resources, Cat. No. NR-37374).

2.5. Protective efficacy studies in guinea pigs

Vaccinated guinea pigs were shipped to ABSL4 facility, TBRI, San Antonio, TX. Animals from Group 1 were randomly assigned to JUNV- and MACV-challenged sub-groups. JUNV (Romero) and MACV/Carvallo (TBRI) were inoculated SC at 1 × 103 PFU/0.1 ml per animal. Animals were observed twice per day and body weight and temperature (telemetry) were monitored. The disease progression was assessed using clinical scoring. All guinea pigs in the JUNV-challenged control group became febrile, lost weight and met euthanasia criteria two weeks after challenge. RNA from animal tissue samples was extracted and subjected to qRT/PCR targeting JUNV (Romero) L RNA (GenBank: #AY619640.1). JUNV L RNA was cloned and used to generate a standard curve. No clinical signs of disease were observed in any of the MACV/TBRI-challenged guinea pigs. The sequence analysis of MACV/TBRI viral RNA revealed several mutations, which presumably appeared during passages of the virus in Vero cells at TBRI (Table S1). The second MACV challenge study was performed with MACV Carvallo from Dr. Paessler (UTMB). Six guinea pigs were immunized with JV&MV GPC VLPV vaccine (prime-boost) as previously described and 5 animals were immunized with Candid #1 (1 × 103 PFU, SC). In the mock-vaccinated group, all animals were morbid and febrile 9 days after infection. Guinea pigs gradually lost weight and met euthanasia criteria at the end of week 3 after challenge. RNAscope analysis documented specific MACV RNA sequences in tissues of these animals using a target probe to MACV Carvallo NP (GenBank #AY619643.1, nt 1861–2869) (Fig. S1).

2.6. RNA sample preparation and deep sequencing

MACV viral RNA was extracted with TRIzol LS reagent (Invitrogen, Grand Island, NY, USA) and subjected to deep sequences [48]. RNA Clean up steps were performed to increase sequence depth. An Illumina TruSeq total RNA sample preparation kit (Illumina, Inc., San Diego, CA, USA) was used to prepare RNA libraries for sequencing. An Illumina pipeline was used to generate FASTQ files for downstream analyses. Files containing all sequence reads were mapped to reference sequence (MACV Carvallo, S and L RNA segments GenBank #AY129248 and AY358021, respectively) using Lasergene SeqMan NGen (DNASTAR, Madison, WI, USA).

2.7. Statistical analysis

Results are reported as means ± SEM (n = 4–5). ANOVA with Bonferroni’s post-hoc test (for parametric data) or Mann-Whitney Rank Sum test (for nonparametric data) was used for the determination of statistical significance among treatment groups, as appropriate. Statistical analysis (mean, SD, T-test) and graphics were performed using the OriginLab 2016 package (OriginLab Corporation, Northampton, MA).

3. Results

3.1. Design of bivalent VLPVs simultaneously expressing JUNV and MACV GPCs specifically stained with monoclonal and polyclonal Abs

We have used the VEEV TC-83 genetic backbone to design bi-cistronic VLPVs expressing codon-optimized GPCs from JUNV and MACV [40]. Cross-reactivity between JUNV and MACV represented an obstacle for the development of an identity assay [49]. Analysis of the crystal structure of MACV GP1 in complex with TfR1 (CD71) revealed three loops, L3, L7 and L10, which protrude from the GP1 core to bind to their receptor [28]. Among them, the MACV L10 is unique because of its longer sequence. To raise specific Ab recognizing MACV GP1, two peptides derived from N and C terminal ends of the L10, peptide Y214-C229 and H233-L248, were synthesized and used for rabbit immunization. The KLH-conjugated peptide H233-L248 had low immunogenicity. The peptide Y214-C229 with amino acid residue F226, which is essential for binding to human TfR1 and cell entry [50], was more immunogenic. Ab against KLH-conjugated peptide Y214-C229 was further antigen-affinity purified and used for identity assay in IFA.

To test specificity of MACV GP1214–229 Ab, Vero cells were infected either with Candid #1 or with bivalent VLPVs and stained with mAb against JUNV and with affinity-purified MACV GP1214–229 Ab. As an additional positive control, BSC-40 cells infected with rVACV-MACV GPC were also used. As expected, cells infected with Candid #1 were positively stained for JUNV GPC with specific clone LD05-BF09 (Fig. 1, panels B, green staining). Notably, JUNV-cells did not cross-react with Ab against MACV GP1 and were stained only with DAPI. Meanwhile, cells infected with JV&MV VLPVs were replicon-specifically stained with either Ab for JUNV GPC (panels C, green), or with Ab for MACV GP1 (panels C, red), and were co-stained with both Abs (panels C, yellow areas indicated by arrows). BSC-40 cells infected with rVACV/MACV GPC were also positively stained with MACV GP1214–229 Ab (not shown). Cells infected with bivalent JV&MV VLPVs expressed roughly equal amount of GPC stained with mAb LD05-BF09 or with MACV GP1214–229 Ab as assessed by flow-cytometry (Fig. 1, panels D). In summary, the MACV GP1214–229-KLH conjugate induced Ab which was able to specifically stain MACV GP1 and did not cross-react with JUNV GPC.

3.2. Immunogenicity studies in guinea pigs

Immunization with alphavirus encapsulated replicons requires a relatively high dose of VLPVs and a prime-boost strategy. In this study, we used an immunization protocol previously optimized in mice and guinea pigs [41, 51]. The animals were vaccinated SC with 1 × 107 FFU and boosted at D28 with the same dose of VLPVs (Fig. 2A). Immunization did not induce any clinical symptoms or hematological (red, white, and platelet blood cell counts, hemoglobin, hematocrit) abnormalities in experimental animals (not shown). Blood samples were tested in PRNT, IFA, and IgG ELISA with JUNV and MACV antigens (Fig. 2). Low-titers of nAbs were detectable in PRNT with JUNV (Candid #1) as early as 2 weeks after immunization (Fig. 2B). Boost-immunization strongly induced nAb responses measured by PRNT, PCR-NA, and by IFA (Fig. 2B–D). Due to limited access to BSL4 facility, nAbs against MACV were not measured. Nevertheless, guinea pigs vaccinated with JV&MV VLPVs induced humoral responses measured in IFA and in IgG ELISA (Fig. 2D–E). Among all vaccinated guinea pigs, only one animal, GP #32683, did not seroconvert and was not protected against fatal disease in challenge experiments.

3.3. Protective efficacy of JV&MV VLPVs in JUNV (Romero) challenge experiments

Independently of their genetic background, guinea pigs have been widely used as an experimental model of human JUNV infection [32, 52, 53]. In this study, Hartley guinea pigs vaccinated with JN&MV VLPVs were challenged with the highly pathogenic Romero strain (LD50 <1 PFU) to assess the vaccine protection. As seen in Fig. 3, mock-vaccinated animals developed fever on D7 and weight loss on D8 after JUNV infection. Clinical symptoms aggravated, animals became less active, lethargic, and some of them showed signs of hind-limb paralysis on D11. At D14, all infected guinea pigs met euthanasia criteria and were humanely euthanized. At necropsy, predominant pathological lesions included liver necrosis and steatosis, necrosis and cell depletion in the spleen with edema and hemorrhage in lungs (Table S2). The high viral RNA load was detected in tissues (Fig. 3E).

Fig. 3.

Fig. 3.

Fig. 3.

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JUNV challenge of JV&MV VLPV-vaccinated guinea pigs. Immunized animals were challenged with Romero strain of JUNV as described in Materials and Methods. Challenged animals were observed for euthanasia criteria (survival), panel (A); weight loss, panel (B); temperature, panel (C); clinical score, panel (D); and viral RNA load in tissues, panel (E). Clinical manifestations of the disease were assessed using activity/movement scoring: 0, normal posture/inquisitive/exploring the cage; 1, not inquisitive/not exploring cage/ few postural changes/hides some of the time; 2, head down, not inquisitive/not exploring cage, few postural changes, hides most of the time, showing signs malaise such as fever; 3, animal has little to no activity or movement, showing hind-limb paralysis (requires euthanasia), weight loss exceeding 20%. Tissue samples collected at necropsy were used for qRT/PCR targeting JUNV (Romero) L RNA (GenBank, AY619640.1). Arrows in panel (E) indicate viral RNA copies in tissues of seronegative animal #32683 which had high clinical scores, met euthanasia criteria, and had histological alterations in tissues (Table S2).

After challenge, all Candid #1-vaccinated guinea pigs survived and had no clinical manifestations. Histological analysis revealed normal tissue structure with minimal changes in examined organs. In line with clinical and histological observations, JUNV RNA was only barely detectable in the spleen tissue. Likewise, all guinea pigs successfully vaccinated with experimental JV&MV VLPV vaccine also survived JUNV challenge. Guinea pig #32683 developed clinical signs of acute wild-type JUNV infection and met euthanized criteria on D13. Tissues of GP #3268 had high viral load and histology scores (Fig. 3E, Table S2). In contrast, all seroconverted and protected animals had normal or minimal histological changes. A single animal in this group had moderately severe lung congestion. However, it seems that this congestion was not associated with JUNV infection since viral load was not detectable in the lung tissues. JUNV RNA was barely detectable only in the spleen tissues of JV&MV VLPV-vaccinated guinea pigs.

3.4. Challenge with MACV (Carvallo)

In contrast to JUNV (Romero), infection of guinea pigs with the MACV (Carvallo) results in lethality ranging from 0% to 80–100% [33, 53–56]. Infection of guinea pigs with 1 × 103 PFU of Carvallo/TBRI did not induce clinical signs of the disease. Viral RNA isolated from this stock had five mutations in the L gene (Table S1). Two non-synonymous substitutions resulted in amino acid changes at position 166 and 781, G>E and M>I, respectively. One synonymous mutation was detected in S RNA encoding NP protein in 28.1% of reads. Association of these mutations with MACV attenuation is not clear and has to be further investigated. Genomic sequence of attenuated MACV Carvallo/TBRI was submitted in GenBank (##MT015969, MT01597).

A second challenge experiment was performed with the UTMB stock of MACV (Carvallo). After challenge, all mock-vaccinated animals had clinical manifestations, begin to lose body weight, appetite, and became febrile between days 8–10 after infection. Animals became passive, lethargic, and expressed neurological signs starting with hind-limb paralysis. At D20, all infected animals met euthanasia criteria. At necropsy, histological changes in examined tissues included portal and parental necrosis in liver, steatosis, spleen lymphoid depletion, lung inflammation and edema (Table S3). In situ RNAScope with MACV (Carvallo) probe targeting NP gene detected specific signals in liver (Fig. S1). JV&MV VLPV-vaccinated animals had no clinical signs of the disease and gained body weight. Histology revealed normal or minimal-mild alterations in tissues (Table S3). All animals were also fully protected against fatal disease in the Candid #1-vaccinated group. These guinea pigs also expressed rapid gain in body weight after D9. The temperature of Candid #1-vaccinated animals remained in normal range during 20 days, dropped at D21–22 and returned to normal range at the end of observation period (Fig. 4). Histological examinations did not reveal pathological changes in tissues. In situ hybridization did not detect MACV-specific signals in tissues of protected guinea pigs (not shown).

Fig. 4.

Fig. 4.

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MACV challenge of JV&MV VLPV-vaccinated guinea pigs. Immunized animals were challenged with Carvallo strain of MACV (UTMB challenge stock) as described in Materials and Methods. Challenged animals were observed for euthanasia criteria (survival), panel (A); weight loss, panel (B); temperature, panel (C); and clinical score, panel (D). Clinical scoring was performed as described in the legend to Fig. 4.

4. Discussion

Since 2007, when Candid #1 vaccine was included in the National Immunization program to vaccine individuals at the age 15 years and older, the incidence of AHF has been substantially reduced [25]. Candid #1 continues to be a cost-effective, single-shot solution, with a relatively low manufacturing cost, and high immunogenicity [8, 25]. Updated epidemiological data indicate an increase in the incidence of AHF in children living in endemic areas [9]. Additionally, an increase in the percentage of females and residents of urban areas among confirmed AHF cases was noted [57]. As with any live-attenuated vaccine, Candid #1 is vulnerable for reversion to a wild-type phenotype. Since limited GP2 missense mutations are the only driving mechanism of Candid #1 attenuation, this reversion potential is becoming a concern for this vaccine. In attempts to design a safer vaccine with broader coverage, Seregin et al. [24] used alphavirus VEEV TC-83-based replicon particles expressing Candid #1 GPC. The authors documented that prime-boost immunization induced full protection against lethal challenge providing evidence that an immune response against GPC alone was sufficient for full protection. Notably, brain tissues of protected animals were virus-free and protected guinea pigs did not develop late neurological manifestations.

We pioneered the application of alphavirus VLPVs based on attenuated VEEV for design of bi-cistronic and multivalent experimental arenavirus vaccines [38, 40, 41, 51] since alphavirus replicon platform provides safe and immunogenic solution for vaccine development. A key safety feature is based on the limited replication of VLPV vaccines in targeted cells. The ability to specifically target DCs and macrophages, as well as high levels of expression of foreign genes under control of 26S promoters, drive the high immunogenicity of alphavirus replicon-based vaccines. Notably, “empty” VLPVs alone (without a transgene,) formulated as an adjuvant, dramatically improved the immunogenicity and protective efficacy of an inactivated influenza Fluzone® vaccine [58]. In the present study, we tested bi-cistronic VLPVs simultaneously expressing GPC of JUNV and MACV in guinea pigs challenge model. Although JUNV and MACV are genetically closely-related species, Abs against JUNV did not neutralize MACV [33–35]. Likewise, anti-MACV GPC nAbs were highly specific to MACV with little cross-reactivity to JUNV [36]. Structural analysis revealed that loop L10 is unique for MACV in terms of sequence and length [28] and restricts accessibility of RBS for heterologous Abs, including closely-related Abs against JUNV GPC [29, 34]. The Y214-C229 peptide derived from MACV L10 was selected to raise Ab against MACV GP1. This Ab specifically interacted with MACV GPC and did not cross react with JUNV GPC. Staining of cells infected with bi-cistronic JV&MV VLPVs with affinity-purified MACV GP1214–229 Ab and with mAb specific for JUNV GP1 provided evidence of simultaneous expression of JUNV and MACV GPCs in the same cells (Fig. 1C). In line with Segerin et al. [24], a second immunization with VLPVs efficiently boosted humoral responses against JUNV assessed in PRNT and in PCR-NA (Fig. 2B, C). Likewise, IFA and IgG ELISA documented induction of MACV-specific Abs after prime-boost vaccination of guinea pigs with bivalent JV&MV VLPVs (Fig. 2D, E). Notably, Candid #1-induced Abs cross-reacted with gamma-irradiated MACV in IgG ELISA (Fig. 2Di).

All guinea pigs that seroconverted after vaccination with JV&MV VLPVs were fully protected against fatal AHF (Fig. 3A). Of 11 VLPV-vaccinated animals enrolled in the first challenge protocol, only GP #32683 did not seroconvert. Since all animals enrolled in the second challenge protocol (with MACV/Carvallo from UTMB) also developed virus-specific Abs, failure to immunize GP #32683 seemed to be a technician error. As expected, GP #32683 was not protected against fatal disease. Likewise, mock-vaccinated guinea pigs had high clinical scores, lost weight and met euthanasia criteria. At necropsy, high viral RNA load and histology grades were seen in tested tissues of mock-vaccinated guinea pigs and in GP #32683 (Fig 3E, Table S2). As expected, Candid #1-vaccinated animals effectively controlled JUNV replication in tissues and were fully protected against fatal AHF. Nevertheless, vaccination with either JV&MV VLPVs or Candid #1, did not induce sterilizing immunity as there were barely detectable levels of JUNV RNA in spleen tissues (Fig. 3E).

Early attempts to develop attenuated MACV vaccine via Candid #1-like cell-culture adaptation approach failed. Stable attenuation was not observed even after 60 passages [53]. Inactivated MACV vaccine was only marginally successful [53]. Recent genetic studies revealed that the F438I substitution in the transmembrane domain of MACV GP2 resulted in attenuated MACV replication in IFNα/β/γR−/− (AG129) mice. However, this attenuation was unstable in vivo [20]. Recombinant MACV expressing Candid #1 GPC (rMACV/Cd#1-GPC) was more genetically stable and induced protection against MACV challenge in AG129 mice [59]. Despite the susceptibility of AG129 and STAT-1 mice to MACV infection, these immunocompromised mice do not comply with the FDA Animal Rule [60].

Information on MACV infection in guinea pigs is limited and controversial. Susceptibility of these animals to MACV varies significantly and depends on genetic background, passage history, and route of inoculation [33, 53–56]. In the 1st efficacy study, we used a challenge stock of Carvallo strain of MACV prepared at TBRI. Surprisingly, this virus was fully attenuated in Hartley outbred guinea pigs. Deep sequencing revealed two non-synonymous mutation in the L protein, G166E and M781I. Association of these mutations with MACV attenuation needs to be further investigated. In a separate study [56], a glycine-glutamine mutation at position 166 in MACV RNA polymerase resulted in 8-fold decrease in polymerase activity assessed with a mini-genome replicon system. Likewise, Golden et al. [31] reported that after a few passages in Vero cells, the Carvallo strain lost pathogenicity in guinea pigs, and this phenotype was associated with genetic changes in the L RNA segment. Meanwhile, five spleen-to-spleen Carvallo passages in guinea pigs resulted in uniform mortality in these animals [53]. Interestingly, the Chicava strain, isolated in 1994 from a fatal human case, does not require adaptation to guinea pigs and was lethal after aerosol application of 100 PFU [54]. Without adaptation to guinea pigs, Carvallo strain even at doses 10,000-fold higher was not pathogenic in this model [56]. In the 2nd challenge study, we used a Carvallo stock from Dr. Paessler’s lab (UTMB). This stock has the prototypic consensus sequence (GenBank ##AY12948, AY358021) and was highly pathogenic for Hartley guinea pigs as assessed by clinical manifestations, weight loss, MACV-specific histology changes in target tissues and detection of MACV RNA in situ by RNAscope hybridization (Fig. 4, S1, Table S3). The experimental JV&MV VLPV vaccine and Candid #1 completely protected guinea pigs against MACV challenge (Fig. 4). Protected animals had no clinical signs of the disease, gained weight and had normal or minimal/moderate microscopic changes in tissues (Table S3).

Despite high structural homology between the GPC of JUNV and MACV, anti-JUNV GPC Abs poorly neutralize MACV since MACV GP-1 L10 insert restricts its GP1 RBS accessibility to cross-reactive Abs [33–35]. Nevertheless, in line with anecdotal evidence [53], Candid #1 vaccination protected guinea pigs against fatal MACV infection (Fig. 4). It suggests that JUNV-induced Abs can target other sites of MACV GPC outside of its Tyr211TfR1 pocket. Indeed, at least one mAb generated from B memory cells of a Candid #1 vaccinee demonstrated a weak activity in PRNT with infectious MACV (Carvallo) [61]. Crystal structural analysis of MACV GP1 in complex with the Fab fragment derived from this Ab demonstrated that in vitro cross-neutralization can be achieved by avoiding interaction with MACV GP1 L10 insert. In addition, recent studies documented the contribution of Abs with Fc-effector functions (e.g., ADCC) in protection against LASV and MACV in guinea pig models [36, 62]. Applicability of this mechanism to cross-protection in Candid #1-immunized NHPs requires further elucidation.

In summary, JV&MV VLPVs offer a safer alternative to the Candid #1 vaccine because alphavirus VLPVs are replication-restricted vehicles with highly efficient expression of foreign antigens in targeted cells (DCs, macrophages). Co-expression of JUNV GPC and MACV GPC in these cells can potentially trigger antigen cross-presentation as we previously described [41], and which will be an additional immunological advantage for this kind of vaccines. Prime-boost application of bivalent JV&MV VLPV vaccine induced immune responses which resulted in complete protection in a validated small animal model meeting the FDA Animal Rule requirements. Full protection in guinea pigs provides a reasonable justification for testing this vaccine in available NHP models for experimental AHF and BHF. The well-defined endemic areas of AHF and BHF suggest that prime-boost regiment is realistic and feasible for populations at risk.

Supplementary Material

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Acknowledgments

The authors sincerely thank Dr. Slobodan Paessler (University of Texas Medical Branch, Galveston, TX) for providing Candid #1 and challenge stock of Machupo (Carvallo) virus. These studies were supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under award number R01 AI093450. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Footnotes

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Supplementary Materials

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NIHMS1567427-supplement-1.docx (26.2KB, docx)
2
NIHMS1567427-supplement-2.pptx (2.2MB, pptx)

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