Lassa Fever Virus 11620 Disease is associated with destruction of antigen specific cytotoxic T lymphocytes (CTLs), causing leukopenia, decreased hemoglobin concentration, elevation of liver aminotransferases, and liver lipidosis. Pathogenesis involves initial replication at the site of infection. Lymph node, lung, and other parenchymal organs are important sites of viral growth. Interstitial infiltrates and edema occur during infection, and the macrophage is early and prominently involved. Many epithelial structures contain antigen and nucleic acids, with widespread marginal zone infection and splenic lymphoid follicle necrosis are common findings (Botten et al., 2006). Lassa fever Detectable neutralizing antibodies are absent during acute Lassa virus infection. Low-titer neutralizing antibodies may appear several weeks to months after the resolution of infection. Treatment of LF patients with immune plasma does not protect against disease. These observations strongly suggest that antibody response is not important, instead, cell-mediated immunity is critical for protection against LASV infection in humans. LASV-specific CD4+ T cells have been found in convalescent patients, and CD8+ T-cell response is the main determinant responsible for providing protection against LASV infection (Botten et al., 2006). The natural host is a small African rat, Mastomys natalensis (Fisher-Hoch et al., 2001). Lassa fever is a viral haemorrhagic fever caused by an arenavirus. Arenaviruses produce mostly silent, persistent infection in rodents, and their origin is thought to date back to the evolution of different rodent species, perhaps as much as 9 million years ago. Accidental human exposure to the virus is frequent. Lassa fever begins insidiously, after an incubation period of 7–18 days, with fever, weakness, malaise, severe headache, and a very painful sore throat. Up to a third of hospitalized Lassa fever patients progress to a prostrating illness 6–8 days after onset of fever, usually with persistent vomiting and diarrhea. Bleeding is seen in only 15–20% of patients, limited primarily to the mucosal surfaces or occasionally conjunctival haemorrhages or gastrointestinal or vaginal bleeding. Severe pulmonary edema and acute respiratory distress syndrome is common in fatal cases with gross head and neck edema and hypovolemic shock. Lassa fever has considerable long-term sequelae, in that nearly 30% of patients with Lassa fever infection suffer an acute loss of hearing in one or both ears. About half of these patients show a near or complete recovery by 3–4 months after onset, but the other half continue with significant sensorineural deafness, which after about a year will be permanent (Fisher-Hoch et al., 2001). Baboon Papio cynocephalus 9556 Bank vole Clethrionomys glareolus 447135 Bear Ursus americanus 9643 Birds Passeroidea 175121 Brown Trout Salmo trutta 8032 Buffalo Bison bison 9901 Carnivores Vulpes 9625 Cat Felis catus 9685 Catfishes Siluriformes 7995 Cattle Bos taurus 9913 Chicken Gallus gallus 9031 Chimpanzee Pan troglodytes 9598 chinchillas Chinchillidae 10150 Copper Pheasant Syrmaticus soemmerringii 9067 Deer Cervus elaphus 9860 Deer mouse Peromyscus maniculatus 10042 Dog Canis familiaris 9615 Ducks Anas 8835 Ferret Mustela putorius furo 9669 Fish Hyperotreti 117565 Gerbil Gerbillina 10045 Goat Capra hircus 9925 Gray wolf Canis lupus 9612 Guinea pig Cavia porcellus 10141 Hamster Mesocricetus auratus 10036 Horse Equus caballus 9796 Human Homo sapiens 9606 Macaque Macaca fascicularis 9541 Mongolian Gerbil Meriones unguiculatus 10047 Monkey Platyrrhini 9479 Mouse Mus musculus 10090 None None Parrot Psittacidae 9224 Pig Sus scrofa 9823 Rabbit Oryctolagus cuniculus 9986 Rainbow trout Oncorhynchus mykiss 8022 Rat Rattus 10114 Raven Corvus corax 56781 sei whale Balaenoptera borealis 9768 Sheep Ovis aries 9940 Squirrel Spermophilus richardsonii 37591 Tree shrew Tupaiidae 9393 Trouts, salmons & chars Salmoninae 504568 Turkey Meleagris gallopavo 9103 Vole Microtus ochrogaster 79684 Water buffalo Bubalus bubalis 391902 Lassa fever Virus Ad5 (E1-, E2b-) Vectored Vaccine Recombinant vector vaccine Research Ad5 (E1-, E2b-) Intramuscular injection (i.m.) An adenovirus vectored vaccine encoding lassa virus NP and GPC providing protection in guniea pig model. (Maruyama et al., 2019) The deletion in the structural E1 gene and E2b gene render the vector non-replicative and deletions in the E3 gene allow the vector to be effective even in the presence of pre-existing Ad5 immunity. The LASV GPC or LASV NP were inserted into the Ad5 (E1-, E2b-) vector based platform. The product was then amplified in E.C7 cells, (HEK293 cells) which constitutively express the Ad polymerase and preterminal protein, before undergoing concentration and purification. (Maruyama et al., 2019) Intramuscular injection (i.m.) Lassa Virus NP, GPC (Maruyama et al., 2019) Lassa fever Virus DNA Vaccine encoding GPC gene of LASV (Josiah Strain) United States Army Medical Research Institute of Infectious diseases (USAMRIID) DNA vaccine Research Intradermal injection (i.d.) GPC gene of LASV with post-translational cleavage of GPC into GP1 and GP2 in the host. (Cashman et al., 2017) Intradermal injection (i.d.) Lassa Virus (Josiah Strain) GPC Gene (Cashman et al., 2017) DNA vaccine construction Following exposure to LASV, neutralizing antibody levels increased in the LASV DNA-vaccinated NHP, peaking approximately 21 d post exposure, then declining slightly at the study end point. Initially, white blood cells (WBC) increased in the LASV DNA-vaccinated. Lymphocyte and monocyte populations increased rapidly after exposure in the LASV DNA-vaccinated NHP, stabilizing by day 21. Both hemoglobin and hematocrit increased before becoming stable in the LASV DNA-vaccinated NHP. Platelets increased in the LASV DNA-vaccinated NHP. (Cashman et al., 2017) Cynomolgus macaques (NHP) were vaccinated by intradermal (ID) injection of the vaccine followed by ID-electroporation (EP). . Two separate studies were conducted to com- pare 3 or 2 vaccinations at 4-week intervals. (Cashman et al., 2017) To identify virus levels in the blood post-exposure, serum viremia was measured using a standard plaque assay as described. Neither serum viremia nor fever was observed in any of the LASV DNA-vaccinated NHP at any time-point. One NHP had one plaque present at the 1 £ 10¡1 dilution at day 6 which falls below the limit of quantitation for the assay and is considered a false positive. (Cashman et al., 2017) LASV Challenge All LASV-GPC DNA-vaccinated NHP, regardless of dose group, showed no signs of infection after exposure and survived to the study end point. Final morbidity scores were assigned at the study end point. A score of zero indicated the macaque was well; showing no outward signs of disease; whereas, a score of 10 indicated the NHP was severely ill and met euthanasia criteria. The LASV DNA-vaccinated NHP remained at zero on the morbidity scale for the duration of the study. (Cashman et al., 2017) Lassa fever virus recombinant vector vaccine V-LSG encoding the glycoprotein VO_0004378 Recombinant vector vaccine Research NYBH strains of vaccinia virus [Ref128:Fisher-Hoch et al., 2000] Intramuscular injection (i.m.) Intramuscular injection (i.m.) Recombinant vector construction Vector NYBH strains of vaccinia virus expressed the uncleaved, full-length glycoprotein (Fisher-Hoch et al., 2000). The protection of our animals by Lassa virus glycoprotein expressed in vaccinia virus in the face of a low antibody response supports that antibody alone does not provide protection. Proper folding of LCMV glycoprotein is critical for induction of the humoral response and vaccinia virus-expressed glycoproteins are not posttranslationally processed or transported correctly to the membrane. Thus, the minimal measurable antibody response to glycoprotein after vaccination may be related to improperly folded glycoprotein (Fisher-Hoch et al., 2000). VO_0000286 8 of 9 V-LSG-vaccinated animals survived Lassa virus challenge (Fisher-Hoch et al., 2000). Lassa fever virus recombinant vector vaccine V-LSG/N encoding the glycoprotein and the nucleoprotein VO_0004379 Recombinant vector vaccine Research NYBH strains of vaccinia virus [Ref128:Fisher-Hoch et al., 2000] Intramuscular injection (i.m.) Intramuscular injection (i.m.) Recombinant vector construction Vector NYBH strains of vaccinia virus expressed the nucleoprotein (Fisher-Hoch et al., 2000). Recombinant vector construction Vector NYBH strains of vaccinia virus expressed the glycoprotein (Fisher-Hoch et al., 2000). The protection of our animals by Lassa virus glycoprotein expressed in vaccinia virus in the face of a low antibody response supports that antibody alone does not provide protection. Proper folding of LCMV glycoprotein is critical for induction of the humoral response and vaccinia virus-expressed glycoproteins are not posttranslationally processed or transported correctly to the membrane. Thus, the minimal measurable antibody response to glycoprotein after vaccination may be related to improperly folded glycoprotein (Fisher-Hoch et al., 2000). VO_0000286 9 of 10 monkeys that received the V-LSG/N vaccine survived challenge with Lassa virus (Fisher-Hoch et al., 2000). Lassa fever virus recombinant vector vaccine YFV17D/LASV-GPC encoding the glycoprotein precursor VO_0004380 Recombinant vector vaccine Research Yellow Fever Vaccine 17D (YFV17D) [Ref137:Bredenbeek et al., 2006] Intramuscular injection (i.m.) Intramuscular injection (i.m.) Recombinant vector construction Vector Yellow Fever Vaccine 17D (YFV17D) expressed the Lassa virus glycoprotein precursor (LASV-GPC) (Bredenbeek et al., 2006). The YFV17D/LAS-GPC vaccination elicited humoral immune responses against YFV17D and LASV antigens. Neutralizing antibodies play the major role in protection against yellow fever (Bredenbeek et al., 2006). VO_0000286 A single subcutaneous injection of the recombinant vaccine protected 13 guinea pigs against fatal Lassa Fever (Bredenbeek et al., 2006). Lassa fever virus vaccine ChAdOx1-Lassa-GPC Recombinant vector vaccine Research ChAdOx1 (Chimpanzee adenovirus vector platform) [Ref5730:Fischer et al., 2021] Intramuscular injection (i.m.) Intramuscular injection (i.m.) Josiah Strain Lassa Virus glycoprotein precursor (GPC) (Fischer et al., 2021) Despite conferring 100% protection against clinical disease after LASV challenge, vaccination with ChAdOx1-Lassa-GPC did not induce sterile immunity. Low amounts of LASV RNA were detected in the tissues of immunized animals necropsied on D12 by qRT-PCR. In the lungs, mean differences (±SEM) in viral load between the control animals and prime-boost or prime vaccinates were 6.99 ± 0.68 and 6.90 ± 0.68 log TCID50/g equivalents, respectively. Similarly, mean viral loads in the livers of control animals exceeded that of animals in the prime-boost and prime groups by 5.46 ± 0.68 and 4.83 ± 0.68 log TCID50/g equivalents, respectively. Finally, vaccination with ChAdOx1-Lassa-GPC reduced splenic viral load by 5.94 ± 0.68 and 5.61 ± 0.68 log TCID50/g equivalents after prime-boost or single-dose delivery, respectively. Reductions in viral RNA in the vaccinated animals versus controls were statistically significant for all tissues (p < 0.0001, two-way ANOVA with Tukey’s multiple comparisons test). Meanwhile, no significant differences were observed between the prime-boost and prime immunization groups. Antibody responses specific to LASV nucleoprotein (NP) were mounted in vaccinated and control animals by D12, which further suggested that low-level virus replication occurred (mean ± SEM ELISA titer: prime-boost = 1500 ± 619, prime = 24000 ± 9906, control = 20800 ± 4800). The titer of anti-NP IgG antibodies was significantly lower in animals receiving prime-boost vaccination compared to those receiving a single dose or control vaccination (p = 0.0492, Kruskal–Wallis nonparametric test with Dunn’s multiple comparisons). No viable LASV was isolated from the tissues of any of the animals immunized with ChAdOx1-Lassa-GPC, besides a single lung sample from an animal in the prime vaccination group (2.52 log TCID50/g) at just above the limit of detection of the assay. Meanwhile, infectious virus was detected in all tissues isolated from control animals (mean ± SEM in lung, liver, spleen: 7.58 ± 0.31, 6.77 ± 0.31, 7.15 ± 0.26 log TCID50/g). Differences in mean virus titer between prime-boost vaccinates and controls (lung, liver, spleen: p = 0.0008, 0.0012, 0.0006), as well as between prime vaccinates and controls (lung, liver, spleen: p = 0.0002, 0.0012, 0.0006) were statistically significant (two-way ANOVA with Tukey’s multiple comparisons test). (Fischer et al., 2021) Three groups of animals (n = 10 per group) received the following immunizations: 3.0 × 10^8 IU of ChAdOx1-Lassa-GPC on D-56 and D-28 (prime-boost), 3.0 × 10^8 IU of ChAdOx1-Lassa-GPC on D-28 (prime), or 1.0 × 10^8 IU of ChAdOx1-GFP on D-28 (control). (Fischer et al., 2021) On D12 post challenge, four randomly selected animals from each group were euthanized to perform virological assessments in lung, liver, spleen, and sera. The remaining animals were used to assess survival; animals in the survival cohort were euthanized after meeting humane endpoint criteria or on D-28 post challenge, which marked the study endpoint. All survival cohort guinea pigs vaccinated with ChAdOx1-Lassa-GPC survived challenge and did not develop fevers, experience weight loss, or exhibit other signs of disease. No discernable differences in weight or temperature were observed between prime and prime-boost vaccinates. By contrast, all control animals in the survival cohort developed signs of terminal illness and met humane endpoint criteria (>20% weight loss) on or before D12. The temperatures of control animals began to increase on D3 and animals became febrile by D8. Weight loss was observed by D7 and progressed until euthanasia by D12. (Fischer et al., 2021) On D0, all animals were challenged with 1.0 × 105 TCID50 of GPA-Josiah strain LASV, which was passaged four times in Hartley guinea pigs. GPA LASV harbors a single nucleotide polymorphism in the S genomic segment compared to the wild-type Josiah strain virus. (Fischer et al., 2021) Lassa fever Virus Vaccine MOPEVAC (Modified Mopeia virus expressing antigens of pathogenic arenaviruses) Institut Pasteur Live, attenuated vaccine Research subcutaneous injection subcutaneous injection GPC (Salami et al., 2019) Recombinant vector construction Multiple mutations in the ExoN site of MOPV NP generated a hyperattenuated strain (MOPVExoN6b). MOPVExoN6b was further modified to harbor the envelope glycoproteins of heterologous pathogenic arenaviruses, such as LASV. (Carnec et al., 2018) Blood samples were collected at several time points, and the activation of CD4+ and CD8+ T cells was measured after stimulation with LASV GPC-derived peptides. Our results showed that tumor necrosis factor alpha (TNF-α)-producing CD8+ and CD4+ T cells were induced 14 days after immunization in most immunized animals in response to LASV GPC-derived peptides. In one animal, GPC-specific CD8+ T cells were rather detected 30 days after immunization, while in another one, GPC-specific CD4 T cells circulated for 14 days postimmunization. Importantly, neutralizing antibodies specific for MOPEVACLAS were detected in the plasma of all immunized animals 23 days after immunization. Thus, a single shot of MOPEVACLAS was able to induce both cellular and humoral immune responses against LASV. (Carnec et al., 2018) Four cynomolgus monkeys were immunized with 6 × 10^6 focus-forming units (FFU) 37 days before challenge with LASV (Josiah strain). Three animals were treated with an irrelevant vaccine and used as controls. A single dose of either vaccine was injected during the study. (Carnec et al., 2018) The four animals immunized with MOPEVACLAS survived the challenge. Three animals presented a fever from days 5 to 10, while the temperature of the fourth animal remained within the normal range. No other symptoms were recorded throughout the course of the challenge. Our results showed that MOPEVACLAS fully protects animals against a lethal challenge with LASV. Taken together, these results showed that the hyperattenuated MOPV-based LASV vaccine candidate is effective. (Carnec et al., 2018) Low-grade fever observed for all animals on the day of immunization (Carnec et al., 2018) All animals were then submitted to a challenge with LASV injected subcutaneously (1,500 FFU) (Carnec et al., 2018) Lassa Virus Nucleoprotein Subunit Vaccine VO_0011441 Recombinant vector vaccine Research Vaccinia virus [Ref1163:Morrison et al., 1989]. Intradermal injection (i.d.) Intradermal injection (i.d.) Recombinant vector construction Control animals received either no vaccine or 10^8 PFU of NYBH vaccinia; vaccinated animals received10^8 PFU of V-LSGPC or V-LSN, or a simultaneous injection with both recombinant vaccinia viruses at separate sites, by intradermal injection of 0.1 ml on the back (Morrison et al., 1989). Ninety-four percent of the animals vaccinated with V-LSN survived a Lassa virus challenge in which only 14% of unvaccinated animals and 39% of animals vaccinated with the New York Board of Health (NYBH) strain of vaccinia virus survived (Morrison et al., 1989). Animals were challenged with 10^4 PFU of guinea-pig-passaged Lassa virus by intraperitoneal inoculation 3 to 4 weeks postvaccination (Morrison et al., 1989). licensed Lassa fever human vaccine Generic Unknown VO_0012177 Live, attenuated vaccine Licensed A generic representation of vaccines utilized to prevent Lassa fever in humans, typically employing live, attenuated Lassa virus strains to induce protective immunity. These vaccines aim to mimic natural infection while minimizing the risk of disease. ML29 VO_0004085 Attenuated ML29 is a clone that has been isolated from a Mopeia virus (MOPV) and Lassa virus (LASV) reassortant. It contains the L RNA from MOPV and the S RNA segment from LASV (Lukashevich et al., 2005). Clone ML29 is selected and triple plaque purified. The viruses are grown on Vero E6 cells cultured in Dulbecco's modified minimum Eagle's medium with 2% fetal calf serum, 1% penicillin-streptomycin, and l-glutamine (2 mM) at 37°C in 5% CO2. Cells and virus stocks were free of mycoplasma contamination (Lukashevich et al., 2005). 13 Ten animals were inoculated subcutaneously (s.c.) with the ML29 clone, and 10 guinea pigs received the same dose of MOPV. Eight animals were used as negative controls. At day 30 after vaccination, the animals were s.c. challenged with 10^3 PFU of LASV (Josiah) and followed for 70 days. Liver enzymes were measured in plasma. Vaccinated animals were euthanized on day 70 after challenge, and tissues were removed (Lukashevich et al., 2005). None noted All strain 13 guinea pigs vaccinated with clone ML29 survived at least 70 days after LASV challenge without either disease signs or histological lesions (Lukashevich et al., 2005). In vaccinated animals, LASV infection did not induce alterations in target tissues. The lungs and livers of vaccinated animals looked essentially like normal tissues. There were also no lesions in other major organs (Lukashevich et al., 2005). Infection of strain 13 guinea pigs with MOPV or with the ML29 reassortant was not lethal for the animals and did not induce clinical or biochemical signs of the disease. All animals survived after challenge and had no clinical manifestations. All measured parameters were in normal ranges in ML29-vaccinated guinea pigs. In MOPV-vaccinated animals, a transient elevation of AST and AlkPh in plasma was observed at week 3 after challenge (Lukashevich et al., 2005). Rhesus macaques Two adult rhesus macaques were s.c. injected with 10^3 PFU of ML29. Blood samples were taken weekly and submitted to the clinical laboratory for complete blood counts and standard 20-assay chemistry panels. At days 14 and 28, the monkeys were euthanized and total blood and tissues were collected. A portion of each tissue was submerged in MEM with 10% FCS (for plaque titration) and in RNAlater (for RNA isolation). The remaining tissue portions were fixed in 10% neutral formalin for the preparation of standard histological sections and stained with hematoxylin-eosin (Lukashevich et al., 2005). This data indicates that ML29 vaccination of rhesus macaques results in a short, inapparent, self-limited infection (Lukashevich et al., 2005). The ML29-vaccinated animals were afebrile throughout the experiment and had no clinical manifestations. Hematological and chemical parameters were in the normal ranges, as was gross appearance at necropsy. Detailed histological examination of rhesus macaques infected with the ML29 reassortant revealed no tissue lesions. The ML29 virus replicated poorly in monkeys and was not detectable in the plasma and tissues by conventional infectious plaque assay. The only organ from which the virus was recovered was the spleen. RT/PCR with LASV GPC-derived primers was transiently positive with RNA plasma and tissue samples (Lukashevich et al., 2005). ML29 L-AttV, rLCMV(IGR/S-S) (Mopiea/Lassa reassortant) Profectus Biosciences; University of Texas Medical Branch Reassortant Research Five of the 8 were given ML29 subcuta- neously (s.c.) and 3 were given ML29 intragastrically (i.g.) The Mopeia/Lassa (ML29) reassortant virus used in this study can be an effective, broadly cross-reactive Lassa vaccine. The large (L) genomic segment of ML29 is derived from the mild MOPV. The S segment of ML29 encodes the NP and GP gene products derived from LASV. ML29 appears to be even more attenuated than its parental MOPV both in vitro and in vivo. (Zapata et al., 2013) Five of the 8 were given ML29 subcuta- neously (s.c.) and 3 were given ML29 intragastrically (i.g.) LASV GPC, LASV NP(Salami et al., 2019) Recombinant vector construction ML29 reassortant vaccine expressing the GP and NP of LASV and the Z and L proteins of Mopeia virus (MOPV) (Zapata et al., 2013). Recombinant vector construction ML29 reassortant vaccine expressing the GP and NP of LASV and the Z and L proteins of Mopeia virus (MOPV) (Zapata et al., 2013) SIV-positive animals were classified as slow, median or rapid progressors based on their physical signs and SIV viral loads at set point, i.e. 3 months after infection. Seven of the 8 SIV/ML29 monkeys experienced a drop in SIV titers (median 20%) during the first week after ML29 inoculation, but those titers returned the following week. In both SIV-infected and non-infected controls, showed a decrease in percentage of circulating NK (CD16+) cells a week after vaccination (Figure 5A and B). The CD14+ (monocyte) population showed a modest increase in the SIV-infected group and a marked increase in the control group one week after ML29 vaccination.Anti-LASV IgG antibodies were detected by ELISA from weeks 1 to week 5 and then monthly until the time of death. All animals showed good titers of anti-Lassa IgG except for those 3 given ML29 by the i.g. route. Seven of the 8 SIV-infected vaccines had vigorous ML29-specific cell-mediated immunity by the first week after vaccination. (Zapata et al., 2013) Eight macaques were inoculated with an attenuated LASV vaccine ML-29, in order to determine whether they could still elicit LASV- specific immune responses without developing signs of arenavirus disease. Five of the 8 were given ML29 subcutaneously (s.c.) and 3 were given ML29 intragastrically (i.g.). (Zapata et al., 2013) Two rapid-progressors were euthanized on days 34 and 63 and two median progressors were euthanized on days 57 and 105 after ML29 vaccination (Table 1, Figures 2 and 3). The first euthanized animal (SIV/ML- 3) had high SIV loads and wasted appearance prior to ML29 vaccination. This animal developed a barely- detectable ML29 viremia (103 pfu/ml of plasma) 3 weeks after vaccination. (This titer of 103 pfu ML29/ml is still below the >104 pfu/ml considered to be a disease sign related to poor prognosis in LHF). Transient ML29 viremia (80 pfu/ml of plasma) was also detected one week after vaccination in the second euthanized monkey (SIV/ML-1), and in a long-term surviving monkey (SIV/ ML-8 had 20 pfu/ml plasma) 3 weeks after vaccination. (Zapata et al., 2013) 93 days after infection with SIVmac251, eleven rhesus macaques were enrolled in this study. (Zapata et al., 2013) V-LSG VO_0004080 Vaccina virus V-LSG is a vaccinia viruses expressing the S-segment Lassa glycoprotein (Fisher-Hoch et al., 2000). The sequence is derived from the Josiah strain of Lassa virus, isolated from a patient in Sierra Leone (Fisher-Hoch et al., 2000). Rhesus and Cynomolgus All animals received a single vaccination consisting of 0.2 ml of vaccine given intradermally and simultaneously at four separate sites (each forearm and the lateral aspect of each thigh) at a dilution which delivered to each animal a total dose of 10^9 PFU. All animals were challenged subcutaneously with 10^3 to 10^4 PFU of the Josiah strain of Lassa virus in 0.5 ml of phosphate-buffered saline within 36 to 700 days (Fisher-Hoch et al., 2000). The latest day on which virus could be recovered from serum was day 14, and that from tissues was day 21. Evidence for persistence elsewhere in tissues or fluids in survivors could not be found by cocultivation of tissues taken up to 112 days following challenge. However, autopsy and biopsy material examined by RT-PCR revealed that viral RNA could be detected at least 112 days after challenge (Fisher-Hoch et al., 2000). The response to glycoprotein protects these animals from induction of fatal processes leading to disease and death (Fisher-Hoch et al., 2000). Animals showed little or no disturbance of liver function, even in the face of viremia (Fisher-Hoch et al., 2000). 8 of 9 vaccinated animals survived. This protection against death was significant when compared with the death rate of the controls. These animals showed significantly diminished mean virus titers compared with those of unvaccinated animals. The one V-LSG-vaccinated animal that died reached maximum viremia on day 8, and the last day on which virus was detected in serum was day 11. The animal the died was the one with the longest vaccine-to-challenge interval. Survival diminished as the vaccine-to-challenge interval increased. A trend towards increasing duration of viremia was also observed with increased intervals between vaccination and challenge (Fisher-Hoch et al., 2000). V-LSG + V-LSN VO_0004082 Vaccina virus V-LSG is a vaccinia viruses expressing the S-segment Lassa glycoprotein and V-LSN is a vaccinia viruses expressing the S-segment Lassa nucleoprotein. V-LSG + V-LSN is a vaccine that includes injections of each V-LSG and V-LSN separately (Fisher-Hoch et al., 2000). Each sequence is derived from the Josiah strain of Lassa virus, isolated from a patient in Sierra Leone (Fisher-Hoch et al., 2000). Rhesus and Cynomolgus All animals received a single vaccination consisting of 0.2 ml of vaccine given intradermally and simultaneously at four separate sites (each forearm and the lateral aspect of each thigh) at a dilution which delivered to each animal a total dose of 10^9 PFU. All animals were challenged subcutaneously with 10^3 to 10^4 PFU of the Josiah strain of Lassa virus in 0.5 ml of phosphate-buffered saline within 36 to 700 days (Fisher-Hoch et al., 2000). The latest day on which virus could be recovered from serum was day 14, and that from tissues was day 21. Evidence for persistence elsewhere in tissues or fluids in survivors could not be found by cocultivation of tissues taken up to 112 days following challenge. However, autopsy and biopsy material examined by RT-PCR revealed that viral RNA could be detected at least 112 days after challenge (Fisher-Hoch et al., 2000). When used together, V-LSG and V-LSN can provide sufficient means for protection against Lassa fever (Fisher-Hoch et al., 2000). None noted 5 out of 6 of the monkeys survived. This protection against death was significant when compared with the death rate of the controls. These animals showed significantly diminished mean virus titers compared with those of unvaccinated animals. The animal the died was the one with the longest vaccine-to-challenge interval. Survival diminished as the vaccine-to-challenge interval increased. A trend towards increasing duration of viremia was also observed with increased intervals between vaccination and challenge (Fisher-Hoch et al., 2000). V-LSG/N VO_0004083 Vaccina virus V-LSG/N is a vaccinia viruses expressing the full length glycoprotein and nucleoprotein in the same construct (Fisher-Hoch et al., 2000). V-LSG is a vaccinia viruses expressing the S-segment Lassa glycoprotein (Fisher-Hoch et al., 2000). Rhesus Two animals were vaccinated. Each animal received a single vaccination consisting of 0.2 ml of vaccine given intradermally and simultaneously at four separate sites (each forearm and the lateral aspect of each thigh) at a dilution which delivered to each animal a total dose of 10^9 PFU. All animals were challenged subcutaneously with 10^3 to 10^4 PFU of the Josiah strain of Lassa virus in 0.5 ml of phosphate-buffered saline within 36 to 700 days The latest day on which virus could be recovered from serum was day 14, and that from tissues was day 21. Evidence for persistence elsewhere in tissues or fluids in survivors could not be found by cocultivation of tissues taken up to 112 days following challenge. However, autopsy and biopsy material examined by RT-PCR revealed that viral RNA could be detected at least 112 days after challenge (Fisher-Hoch et al., 2000). The results of this experiment show that V-LSG/N provides effective means of protecting monkeys against the Lassa virus (Fisher-Hoch et al., 2000). None noted Both of the animals survived when challenged with the Lassa virus. These animals showed no significant mean virus titers when compared with those of unvaccinated animals (Fisher-Hoch et al., 2000). V-LSG1 + V- LSG2 VO_0004081 Vaccina virus V-LSG1 + V- LSG2 is a vaccinia viruses expressing the S-segment Lassa single glycoproteins V-LSG1 [containing residues 1 to 296] and V-LSG2 [with a deletion of residues 67 to 234] (Fisher-Hoch et al., 2000). The sequence is derived from the Josiah strain of Lassa virus, isolated from a patient in Sierra Leone (Fisher-Hoch et al., 2000). Rhesus All animals received a single vaccination consisting of 0.2 ml of vaccine given intradermally and simultaneously at four separate sites (each forearm and the lateral aspect of each thigh) at a dilution which delivered to each animal a total dose of 10^9 PFU. Each vaccine was administered in one arm and one leg on the ipsilateral side. All animals were challenged subcutaneously with 10^3 to 10^4 PFU of the Josiah strain of Lassa virus in 0.5 ml of phosphate-buffered saline within 36 to 700 days (Fisher-Hoch et al., 2000). The latest day on which virus could be recovered from serum was day 14, and that from tissues was day 21. Evidence for persistence elsewhere in tissues or fluids in survivors could not be found by cocultivation of tissues taken up to 112 days following challenge. However, autopsy and biopsy material examined by RT-PCR revealed that viral RNA could be detected at least 112 days after challenge (Fisher-Hoch et al., 2000). Although V-LSG1 and V-LSG2 do not provide sufficient protection by themselves, when used together the can provide protective immunity to primates. This means that both glycoproteins are independently important in Lassa virus protection (Fisher-Hoch et al., 2000). None noted Both of the monkeys vaccinated with V-LSG1 + V-LSG2 were protected when challenged Lassa virus (Fisher-Hoch et al., 2000). V-LSGPC VO_0004086 Vaccina virus V-LSGPC is a cloned cDNA containing the complete glycoprotein gene of the Josiah strain of Lassa virus was inserted into the thymidine kinase (TK) gene of the New York Board of Health (WYETH) strain of vaccinia virus (Auperin et al., 1988). The Lassa virus GPC gene was assembled and ligated into the unique SmaI site of the vaccinia virus expression vector pSCl1. The products of this reaction were transfected into competent Escherichia coli MC1061 cells, and a transformant containing the proper orientation of the Lassa GPC gene was identified by restriction enzyme digestion of plasmid DNA and confirmed by nucleotide sequence analysis (Auperin et al., 1988). Protein Hartley Animals were vaccinated with l0^8 plaque-forming units (PFUs) of V-LSGPC recombinant virus using a single intradermal injection of 0.1 ml on the back. l04 PFUs of guinea of pig-cultured Lassa virus were given by intraperitoneal innoculation 21 days post-vaccination. All surviving animals were completely free of virus in their blood and tissues by 61 days postchallenge, when the experiment was terminated (Auperin et al., 1988). Vaccination with V-LSGPC effectively limited the replication of Lassa virus in comparison to the unvaccinated animals. The ability to construct recombinant vaccinia viruses that express heterologous genes offers great potential for vaccine development (Auperin et al., 1988). Two to three days after vaccination, vesicles developed at the site of inoculation on each animal. The vesicles arising from V-LSGPC were approximately 2-3 mm. All vesicles scabbed and completely healed by 19 days postvaccination. vaccinated animals developed mild symptoms of Lassa fever following challenge. The animals vaccinated with V-LSGPC virus developed low-grade fevers, which began about 8 days post-challenge and lasted approximately 4 days. The unprotected animals, however, developed significantly higher fevers, which persisted until they died (Auperin et al., 1988). 21 days post vaccination the animals were challenged with Lassa virus. All animals that received V-LSGPC recombinant virus survived the lethal Lassa virus challenge (Auperin et al., 1988). V-LSN VO_0004079 Vaccina virus V-LSN is a vaccinia viruses expressing the S-segment Lassa nucleoprotein (Fisher-Hoch et al., 2000). The sequence is derived from the Josiah strain of Lassa virus, isolated from a patient in Sierra Leone (Fisher-Hoch et al., 2000). Rhesus and Cynomolgus All animals received a single vaccination consisting of 0.2 ml of vaccine given intradermally and simultaneously at four separate sites (each forearm and the lateral aspect of each thigh) at a dilution which delivered to each animal a total dose of 10^9 PFU. All animals were challenged subcutaneously with 10^3 to 10^4 PFU of the Josiah strain of Lassa virus in 0.5 ml of phosphate-buffered saline within 36 to 700 days (Fisher-Hoch et al., 2000). The latest day on which virus could be recovered from serum was day 14, and that from tissues was day 21. Evidence for persistence elsewhere in tissues or fluids in survivors could not be found by cocultivation of tissues taken up to 112 days following challenge. However, autopsy and biopsy material examined by RT-PCR revealed that viral RNA could be detected at least 112 days after challenge (Fisher-Hoch et al., 2000). Vaccination using the nucleoprotein may protect primates against a lower challenge dose of Lassa virus, however, the V-LSN vaccine was not significantly protective (Fisher-Hoch et al., 2000). Monkeys had marked lymphopenia and higher mean aspartate aminotransferase values than unvaccinated animals. The V-LSN-vaccinated animals were observed to be sicker and died earlier than unvaccinated animals (Fisher-Hoch et al., 2000). 8 of the 11 vaccinated animals died, which shows that this vaccine was not significantly protective. The V-LSN animals appeared to have a shorter and more acute process than unprotected animals. The median day of death for V-LSN animals was day 11.5, compared with day 13 for the control animals. This phenomenon was related to the challenge dose. Back titration of the challenge inoculum used in the final experiment showed that the titer had dropped from 10^4 to 10^3 PFU/ml. The three V-LSN-vaccinated animals in that experiment that received the lower challenge titer survived. These three animals were rhesus monkeys (Fisher-Hoch et al., 2000). VSV[Delta]G/LVGPC VO_0004087 Live Attenuated VSV[Delta]G/LVGPC ia a live attenuated recombinant vesicular stomatitis virus expressing the GPC of Lassa virus, strain Josiah. Vaccines based on live attenuated rVSV have been highly effective in animal models and are particularly attractive because they can be administered by the mucosal route (Geisbert et al., 2005). The recombinant VSV expressing the glycoprotein of Lassa virus, strain Josiah, and Zaire ebolavirus (ZEBOV), strain Mayinga, were generated using the infectious clone for the VSV, Indiana serotype. Briefly, the appropriate open reading frames for the glycoproteins were generated by PCR, cloned into the VSV genomic vectors lacking the VSV glycoprotein gene, sequenced-confirmed, and rescued. The recombinant viruses expressing Lassa virus glycoprotein and ZEBOV glycoprotein were designated VSV[Delta]G/LVGPC (Figure 1A) and VSV[Delta]G/ZEBOVGP, respectively (Geisbert et al., 2005). Protein Cynomolgus macaques Four cynomolgus macaques, 4–6 y old and weighing between 3 kg and 8 kg, were vaccinated intramuscularly with approximately 2 × 10^7 PFU of VSV[Delta]G/LVGPC, and two with an equivalent dose of VSV[Delta]G/ZEBOGP (controls). The six cynomolgus macaques were challenged intramuscularly 28 d after the single-dose immunization with 1 × 10^4 plaque-forming units of Lassa virus, Josiah strain (Geisbert et al., 2005). None noted The VSV-based vector expressing the Lassa virus GPC mediated complete protection of four of four cynomolgus monkeys from a high-dose lethal challenge of Lassa virus. Protection was associated with the generation of Lassa-specific CD8+ T cell and antibody responses. The primary concern regarding use of the rVSV vaccine platform in humans is related to the fact that this is a replication-competent vaccine, and thus demonstration of safety is of paramount importance (Geisbert et al., 2005). After vaccination, none of the nonhuman primates displayed any signs of clinical symptoms, indicating that the rVSVs were apathogenic for these animals (Geisbert et al., 2005). After the challenge, the two control animals started to show clinical signs of illness on day 3, when one of the animals had a fever. By day 10, both control animals developed macular rashes and anorexia, and one animal had severe facial edema, which is prognostic for a poor outcome in humans. These control animals succumbed to the Lassa virus challenge and were euthanized on day 11 and day 13. At necropsy, both controls showed lesions and pathological changes consistent with Lassa fever in nonhuman primates. In contrast, none of the vaccinated animals became sick, and all four animals were fully protected against the high Lassa challenge dose. By day 7 after challenge, all six monkeys were viremic. However by day 10, all four of the vaccinated animals had cleared the viremia, while both control animals had high viremias, which was maintained until euthanasia (Geisbert et al., 2005). YFV17D/LAS-GPC VO_0004088 Live Attenuated YFV17D/LAS-GPC is a Yellow Fever Vaccine 17D (YFV17D) that has been used as a vector for the Lassa virus glycoprotein precursor (LASV-GPC) resulting in construction of YFV17D/LASV-GPC recombinant virus. The virus is replication-competent and processes the LASV-GPC in cell cultures (Bredenbeek et al., 2006). The YFV17D/LASV-GPC plasmid is constructed in the background of the full-length YFV17D cDNA clone by fusion PCR mutagenesis. The LASV-GPC gene of the AV strain is amplified by RT/PCR and cloned into pcDNA. The nucleotide sequences of PCR-derived DNA fragments and gene fusions are confirmed by sequencing. The recombinant YFV17D/LASV-GPC plasmid is linearized by XhoI and used for in vitro RNA transcription (Bredenbeek et al., 2006). 13 Sixteen outbred guinea pigs were subcutaneously inoculated with 1 × 10^5 PFU/0.5 ml of the recombinant YFV17D/LASV-GPC virus and two animals were sacrificed at days 0, 4, 7, 10 and 14 to track the virus distribution in blood and tissues. At day 14, six animals were boosted with the same dose of the recombinant virus and plasma samples were collected on days 8, 15 and 24 after to measure antibody responses against YFV17D and LASV-GPC in IgG ELISA. Antigens were prepared from serum-free virus stocks of YFV17D and MOP/LAS (Lukashevich et al., 2005) by ultracentrifugation on sucrose cushion. Concentrated viruses were suspended in carbonate–bicarbonate buffer, briefly sonicated and used to cover wells of microtitration plates overnight at 4 °C. After blocking, 1:100 dilutions of guinea pig sera were added and incubated for 2 h at room temperature. Challenge experiments were preformed (Bredenbeek et al., 2006). None noted 80% of animals were protected against the fatal challenge. This study demonstrates the potential to develop an YFV17D-based bivalent vaccine against Lassa Virus (Bredenbeek et al., 2006). None noted 80% of animals were protected against the fatal challenge. Incomplete protection could be explained by differences in vaccine formulation (GPC + NP vs. GPC) and by GPC sequence differences between AV and Josiah strains of LASV. Blood and tissue samples were collected from vaccinated animals at different time points for hematology, blood chemistry, RNA extraction, plaque assay, virus isolation and ELISA. As expected, the inoculated animals had no clinical manifestations and all standard measurable blood and chemistry parameters were in normal ranges. In plasma, the recombinant virus was not detectable by plaque assay or by biological amplification. Recombinant viral RNA was not detectable by RT/PCR in 140 μl of plasma extracted on days 4, 10, and 21. However, when RNA samples were prepared from 0.5 ml of total blood, an RT/PCR assay gave a strong positive signal on day 4 after YFV17D/ LAS-GPC inoculation. Still, blood samples collected on day 10 and 21 were PCR negative. Viral RNA sequences were only transiently detectable on days 7–14 in spleen and liver. Nucleotide sequence analysis of PCR products confirmed their derivation from YFV17D/LAS-GPC. Taken together, these data confirm that recombinant YF17D/LAS-GPC replicated poorly in tissues of vaccinated guinea pigs. Interestingly, in clinical trials in individuals vaccinated with chimeric YFV17D-based vaccines, viremia levels were even lower than the levels of YFV17D determined to be safe. This suggests that insertion of a foreign gene can affect in vivo viral replication and make recombinant YFV17D-based vaccines even safer than the parental vaccine, YFV17D (Bredenbeek et al., 2006). GPC Lassa virus strain AV 354681511 CDD:279177 11620 ? GPC protein 7.81 54591.5 547 Arenavirus glycoprotein; pfam00798 >CCA30314.1 GPC protein [Lassa mammarenavirus] MGQIVTFFQEVPHVIEEVMNIVLIALSILAVLKGLYNIATCGLIGLITFLLLCGRSCSSNLYKGVYELQS LDLNMETLNMTMPLSCTKNNSHHYIRVGNDTGLELTLTNTSILNHKFCNLSDAHKKDLYDHALMSIISTF HLSIPNFNQYEAMSCDFNGGKISVQYNLSHSYAIDAANHCGTVANGILQTFMRMAWGGSYIALDSGRGNW DCIMTSYQYLIIQNTTWENHCQFSRPSPIGYLGLLSQRTRDIYISRRLLGTFTWTLSDSEGNATPGGYCL TRWMLIEAELKCFGNTAVAKCNEKHDEEFCDMLRLFDFNKQAISRLKSEAQMSIQLINKAVNALINDQLI MKNHLRDIMGIPYCNYSKYWYLNHTITGKTSLPKCWLVSNGSYLNETHFSDDIEQQADNMITEMLQKEYM ERQGKTPLGLVDLFVFSTSFYLISIFLRLVKIPTHRHIVGKPCPKPHRLNHMGICSCGLYKQPGVPVRWK R Protective antigen LASVsSgp1 nucleoprotein Lassa mammarenavirus VO_0011151 956584 23343510 LASVsSgp1 AY628203 NP_694869 11620 segment S 100 1809 + nucleoprotein 8.58 59623.51 569 >NC_004296.1:100-1809 Lassa virus segment S, complete sequence GATGAGTGCCTCAAAGGAAATAAAATCCTTTTTGTGGACACAATCTTTGAGGAGGGAATTATCTGGTTAC TGCTCCAACATCAAACTACAGGTGGTGAAAGATGCCCAGGCTCTTTTACATGGACTTGACTTCTCCGAAG TCAGTAATGTTCAACGGTTGATGCGCAAGGAGAGAAGGGATGACAATGATTTGAAACGGTTGAGGGACCT AAATCAAGCGGTCAACAATCTTGTTGAATTAAAATCAACTCAACAAAAGAGTATACTGAGAGTTGGGACT CTAACCTCAGATGACTTATTAATCTTAGCCGCTGATCTAGAGAAGTTAAAGTCAAAGGTGATCAGAACAG AAAGGCCATTAAGTGCAGGTGTCTATATGGGCAACCTAAGCTCACAGCAACTTGACCAAAGAAGAGCTCT CCTGAATATGATAGGAATGAGTGGTGGTAATCAAGGGGCTCGGGCTGGGAGAGATGGAGTGGTGAGAGTT TGGGATGTGAAAAATGCAGAGTTGCTCAATAATCAGTTCGGGACCATGCCAAGTCTGACACTGGCATGTC TGACAAAACAGGGGCAGGTTGACTTGAATGATGCAGTACAAGCATTGACAGATTTGGGTTTGATCTACAC AGCAAAGTATCCCAACACTTCAGACTTAGACAGGCTGACTCAAAGTCATCCCATCCTAAATATGATTGAC ACCAAGAAAAGCTCTTTGAATATCTCAGGTTATAATTTTAGCTTGGGTGCAGCTGTGAAGGCAGGAGCTT GCATGCTGGATGGTGGCAATATGTTGGAGACAATCAAGGTGTCACCTCAGACAATGGATGGTATCCTCAA ATCCATTTTAAAGGTCAAGAAGGCTCTTGGAATGTTCATTTCAGACACCCCTGGTGAAAGGAATCCTTAT GAAAACATACTCTACAAGATTTGTTTGTCAGGAGATGGATGGCCATATATTGCATCAAGAACCTCAATAA CAGGAAGGGCCTGGGAAAACACTGTCGTTGATCTGGAATCAGATGGGAAGCCACAGAAAGCTGACAGCAA CAATTCCAGTAAATCCCTGCAGTCGGCAGGGTTTACCGCTGGGCTTACCTATTCTCAGCTGATGACCCTC AAGGATGCAATGCTGCAACTTGACCCAAATGCTAAGACCTGGATGGACATTGAAGGAAGACCTGAAGATC CAGTGGAAATTGCCCTCTATCAACCAAGTTCAGGCTGCTACATACACTTCTTCCGTGAACCTACTGATTT AAAGCAGTTCAAGCAGGATGCTAAGTACTCACATGGGATTGATGTCACAGACCTCTTCGCTACACAACCG GGCTTGACCAGTGCTGTCATTGATGCACTCCCCCGGAATATGGTCATTACCTGTCAGGGGTCCGATGACA TAAGGAAACTCCTTGAATCACAAGGAAGAAAAGACATTAAACTAATTGATATTGCCCTCAGCAAAACTGA TTCCAGGAAGTATGAAAATGCAGTCTGGGACCAGTATAAAGACTTATGCCACATGCACACAGGTGTCGTT GTTGAAAAGAAGAAAAGAGGCGGTAAAGAGGAAATAACCCCTCACTGTGCACTAATGGACTGCATCATGT TTGATGCAGCAGTGTCAGGAGGACTGAACACATCGGTTTTGAGAGCAGTGCTGCCCAGAGATATGGTGTT CAGAACATCGACACCTAGAGTCGTTCTGTA >NP_694869.1 nucleoprotein [Lassa mammarenavirus] MSASKEIKSFLWTQSLRRELSGYCSNIKLQVVKDAQALLHGLDFSEVSNVQRLMRKERRDDNDLKRLRDL NQAVNNLVELKSTQQKSILRVGTLTSDDLLILAADLEKLKSKVIRTERPLSAGVYMGNLSSQQLDQRRAL LNMIGMSGGNQGARAGRDGVVRVWDVKNAELLNNQFGTMPSLTLACLTKQGQVDLNDAVQALTDLGLIYT AKYPNTSDLDRLTQSHPILNMIDTKKSSLNISGYNFSLGAAVKAGACMLDGGNMLETIKVSPQTMDGILK SILKVKKALGMFISDTPGERNPYENILYKICLSGDGWPYIASRTSITGRAWENTVVDLESDGKPQKADSN NSSKSLQSAGFTAGLTYSQLMTLKDAMLQLDPNAKTWMDIEGRPEDPVEIALYQPSSGCYIHFFREPTDL KQFKQDAKYSHGIDVTDLFATQPGLTSAVIDALPRNMVITCQGSDDIRKLLESQGRKDIKLIDIALSKTD SRKYENAVWDQYKDLCHMHTGVVVEKKKRGGKEEITPHCALMDCIMFDAAVSGGLNTSVLRAVLPRDMVF RTSTPRVVL Protective antigen Vaccine efficacy trials in guinea pigs indicated that the nucleoprotein is capable of eliciting a protective immune response against a lethal dose of Lassa virus. Ninety-four percent of the animals vaccinated with V-LSN survived a Lassa virus challenge in which only 14% of unvaccinated animals and 39% of animals vaccinated with the New York Board of Health (NYBH) strain of vaccinia virus survived [Ref1163:Morrison et al., 1989]. LASVsSgp2 glycoprotein Lassa mammarenavirus VO_0010869 956585 23343511 LASVsSgp2 AY628203 NP_694870 11620 segment S 1871 3346 - glycoprotein 7.63 53529.32 491 >NC_004296.1:1871-3346 Lassa virus segment S, complete sequence CTCATCTCTTCCATTTCACAGGCACACCAGGCTGTTTGTAGAGTCCACAGGAACAAATGCCCATATGATT CAATCTGTGAGGTTTGGGACACGACTTGCCTACAATATGCCTATGAGTTGGTATTTTGACTAGGTGAAGG AAGATGCTAATAAGATAGAAACTTGTACTGAACACAAAGAGGTCAACTAGACCCAATGGTGTCTTCCCCT GCCTCTCCATATACTCCTTCTGTAACATCTCAGTGATCATATTGTCAGCTTGTTGTTCAATATCATCAGA AAAGTGGGTCTCGTTCAAGTATGAACCATTTGATACAAGCCAACATTTGGGCAGTGATGTTCTCCCAGTA GTTGTGTGGTTGAGGTACCAATACTTGCTGTAATTACAGTATGGAATTCCCATGATGTCCCGTAGATGGT TCTTCATTATAAGTTGGTCATTTATCAAAGCATTTACTGCTTTGTTGATCAACTGAATGCTCATTTGTGC TTCAGCTTTCAACCTTTGAATGGCTTGTTTGTTGAAGTCAAACAGCCTCAGCATGTCACAAAATTCCTCA TCATGCTTCTCATTACATTTTGCCACAGCTGTGTTCCCGAAGCATTTTAGTTCAGCCTCAATTAGCATCC ACCTGGTCAGACAATATCCCCCTGGTGTGTCTTTACCTTCAGAATCTGACAGTGTCCATGTGAATGTGCC TAGCAATCTTCTACTAATATAAATATCTCTAGTCCTTTGTGAGAGGAGCCCGAGATAACCGATGGGAGAT GGTCTCGAGAATTGGCAGTGATCTTCCCAGGTTGTATTTTGGATTATCAGATATTGATAACTAGTCATAA TACAGTCCCAGTTGCCACGGCCTGAGTCAAGAGCAATGTAGCTCCCACCCCAAGCCATCCTCATAAAAGT CTGTAACACACCATTTGCAACAGTACCACAATGGTTGGCTGCATCCCCAGCATAGCTGTGACTCAGGTTG TACTGCACACTAATCTTTCCCCCATTAAAATCGCAGCTCATTGCCTCATACTGATTGAAGTTGGGGATGG ACAAGTGGAAAGTTGAGATTATGCTCATAAGAGCGTGGTCATAGAGGTTCTTTTTGTGGGCATCAGACAG ATTGCAAAATTTGTGATTAATAATGCTCGTGTTGGTCAAGGTCAGTTCTAGTCCTGTCTCATTGCCCACC ATTATATAATGATGACTGTTGTTCTTTGTGCAGGAGAGAGGCATGGTCATATTGAGTGTCTCCATGTTTA GTTCCAGAGTCTGAAGCTCATAAACCCCTTTATAAAGACTGGTTGTGCAAGACCTACCACACAACAGGAG GAAAGTGACCAAACCAACAAGGCCACACGTTGCAAAATTGTACAGACCTTTCAGCACTGCTAGTACAGAC AGTGCAATGAGAACAATGTTCATCACCTCTTCTATTACATGAGGCACTTCCTGGAAGAATGTCACTATTT GTCCCA >NP_694870.1 glycoprotein [Lassa mammarenavirus] MGQIVTFFQEVPHVIEEVMNIVLIALSVLAVLKGLYNFATCGLVGLVTFLLLCGRSCTTSLYKGVYELQT LELNMETLNMTMPLSCTKNNSHHYIMVGNETGLELTLTNTSIINHKFCNLSDAHKKNLYDHALMSIISTF HLSIPNFNQYEAMSCDFNGGKISVQYNLSHSYAGDAANHCGTVANGVLQTFMRMAWGGSYIALDSGRGNW DCIMTSYQYLIIQNTTWEDHCQFSRPSPIGYLGLLSQRTRDIYISRRLLGTFTWTLSDSEGKDTPGGYCL TRWMLIEAELKCFGNTAVAKCNEKHDEEFCDMLRLFDFNKQAIQRLKAEAQMSIQLINKAVNALINDQLI MKNHLRDIMGIPYCNYSKYWYLNHTTTGRTSLPKCWLVSNGSYLNETHFSDDIEQQADNMITEMLQKEYM ERQGKTPLGLVDLFVFSTSFYLISIFLHLVKIPTHRHIVGKSCPKPHRLNHMGICSCGLYKQPGVPVKWK R Protective antigen HLA-A*0201 mice immunized with either GPC(42-50) from LASVsSgp2 glycoprotein or GPC(60-68) were protected against challenge with a recombinant vaccinia virus that expressed LASV GPC [Ref132:Botten et al., 2006]. 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