Therapy for Argentine hemorrhagic fever in nonhuman primates with a humanized monoclonal antibody.

The COVID-19 pandemic has reemphasized the need to identify safe and scalable therapeutics to slow or reverse symptoms of disease caused by newly emerging and reemerging viral pathogens. Recent clinical successes of monoclonal antibodies (mAbs) in therapy for viral infections demonstrate that mAbs offer a solution for these emerging biothreats. We have explored this with respect to Junin virus (JUNV), an arenavirus classified as a category A high-priority agent and the causative agent of Argentine hemorrhagic fever (AHF). There are currently no Food and Drug Administration-approved drugs available for preventing or treating AHF, although immune plasma from convalescent patients is used routinely to treat active infections. However, immune plasma is severely limited in quantity, highly variable in quality, and poses significant safety risks including the transmission of transfusion-borne diseases. mAbs offer a highly specific and consistently potent alternative to immune plasma that can be manufactured at large scale. We previously described a chimeric mAb, cJ199, that provided protection in a guinea pig model of AHF. To adapt this mAb to a format more suitable for clinical use, we humanized the mAb (hu199) and evaluated it in a cynomolgus monkey model of AHF with two JUNV isolates, Romero and Espindola. While untreated control animals experienced 100% lethality, all animals treated with hu199 at 6 d postinoculation (dpi) survived, and 50% of animals treated at 8 dpi survived. mAbs like hu199 may offer a safer, scalable, and more reproducible alternative to immune plasma for rare viral diseases that have epidemic potential.

[Sensitivity and specificity of the ELISA Kit for the detection of antidobies to Junin virus].

The goal of this work was to describe methodological approaches to determination of sensitivity and specificity of the enzyme-linked immunosorbent assay kit (ELISA Kit) for detection of the specific anti-Junin virus (JV) antibody. Comparison of ELISA to plaque reduction neutralization test (PRNT) showed direct relationship between antibody titers in the samples of serum of immunized animals, determined by either PRNT or ELISA methods. The obtained results provided an opportunity to form the panels of positive and negative serum samples to determine the sensitivity and specificity of the ELISA Kit. Sensitivity of the ELISA Kit was at least 98% when studying the samples of serum of immunized guinea pigs and rabbits (determined as positive in PRNT). The sensitivity of the ELISA Kit was at least 68% when studying the samples determined by PNRT as uncertain positive. The specificity was 98%. The specificity of the ELISA Kit was 98%.

[EXPERIENCE OF STUDY AND POSSIBLE WAYS OF ELIMINATION OF FALSE POSITIVE AND FALSE NEGATIVE RESULTS DURING EXECUTION OF POLYMERASE CHAIN REACTION ON AN EXAMPLE OF JUNIN VIRUS RNA DETECTION].

AIM: Experience of study and possible ways of elimination of false positive and false negative results during execution of polymerase chain reaction on an example of Junin virus RNA detection. MATERIALSS AND METHODS: Junin virus--causative agent of Argentine hemorrhagic fever (AHF) strain XJpR37/5787 was obtained from the State collection of pathogenicity group I causative agents of the 48th Central Research Institute. Reagent kit for detection of Junin virus RNA by RT-PCR was developed in the Institute and consists of 4 sets: for isolation of RNA, execution of reverse-transcription reaction, execution of PCR and electrophoretic detection of PCR products. RT-PCR was carried out by a standard technique. Continuous cell cultures of African green monkey Vero B, GMK-AH-1(D) were obtained from the museum of cell culture department of the Centre. RESULTS: An experimental study of the effect of various factors of impact on the sample under investigation ("thawing-freezing", presence of formaldehyde, heparin) on the obtaining of false negative results during Junin virus RNA detection by using RT-PCR was studied. Addition of 0.01% heparin to the samples was shown to completely inhibit PCR. Addition of 0.05% formaldehyde significantly reduces sensitivity of the method. A possibility of reduction of analysis timeframe from 15 to 5 days was shown during detection of the causative agent in samples with low concentration of the latter by growing the samples and subsequent analysis of the material obtained by using RT-PCR. CONCLUSION: During detection of causative agent by using RT-PCR false negative results could appear in the presence of formaldehyde and heparin in the sample. A possibility of elimination of false negative PCR results due to concentration of the causative agent in the sample under investigation at a level below sensitivity threshold was shown on the example of Junin virus RNA detection by using growing of the pathogen in appropriate accumulation system with subsequent analysis of the material obtained using PCR.

[Phenotypic markers of attenuation in Junin virus strains recently isolated from individuals vaccinated with Junin Candid#1 strain]. / Marcadores fenotípicos de atenuación en cepas de virus Junín recuperadas de individuos vacunados con la cepa Junín Candid#1.

Argentine hemorrhagic fever is a severe acute disease caused by Junin virus. For prevention of this disease an effective vaccine called Candid#1 has been developed, composed of a live attenuated Junin virus strain. During a clinical trial conducted at Instituto Nacional de Enfermedades Virales Humanas (INEVH) in 2005, Junin virus was isolated from two vaccinated volunteers by co-culture of peripheral mononuclear blood cells. The aim of this study was to compare the strains isolated from these human volunteers with Candid#1 strain regarding phenotypic characteristics of attenuation according to the indicators developed by Contigiani and Sabattini in 1977. The three strains were lethal to suckling mice but not to 10-12 days old mice and guinea pigs. Surviving guinea pigs from primary infection were protected when challenged by intra-muscular inoculation with lethal doses of a virulent strain. Infection and protection rates indicate that these strains are highly infective and protective in the hosts studied herein. These results demonstrate that Junin virus strains isolated from volunteers immunized with Candid#1 maintain the same attenuated phenotype of Candid#1 vaccine after one passage in humans.

Enhanced protection against experimental Junin virus infection through the use of a modified favipiravir loading dose strategy.

A collection of Old and New World arenaviruses are etiologic agents of viral hemorrhagic fever, a syndrome that features hematologic abnormalities, vascular leak, hypovolemia, and multi-organ failure. Treatment is limited to ribavirin for Lassa fever and immune plasma for Argentine hemorrhagic fever. Improved therapeutic options that are safe, more effective and widely available are needed. Here, we show that modification of favipiravir treatment to include a high-dose loading period achieves complete protection in a guinea pig model of Argentine hemorrhagic fever when treatment was initiated two days following challenge with Junin virus (JUNV). This loading dose strategy also protected 50% of animals from lethal disease when treatment was delayed until 5 days post-infection and extended the survival time in those that succumbed. Consistent with the survival data, dramatic reductions in serum and tissue virus loads were observed in animals treated with favipiravir. This is the first report demonstrating complete protection against uniformly lethal JUNV infection in guinea pigs by administration of a small molecule antiviral drug.

Inhibition of cell fusion in Junin virus-infected cells by sera from Argentine hemorrhagic fever patients.

BACKGROUND: Junin virus (JV), a member of the Arenaviridae family, is the etiological agent of Argentine hemorrhagic fever (AHF). A low pH-pulse, induces fusion of Vero cells infected with JV to form syncytia, whose production can be inhibited by neutralizing antibodies against the JV major glycoprotein. OBJECTIVES: To characterize the existence of an antifusogenic activity present in sera obtained from natural infections of AHF over a 20-year period and to study both the fusogenic activity of one pathogenic and two attenuated strains of JV in Vero cells, at different pH. The study sample consisted of sera obtained from two provinces in the Argentine Republic. Vero cells grown in monolayers, were infected with different strains of JV and a 2 h pulse, at different pH, was performed. Syncytium production was evaluated 12 h later, after staining with Giemsa. Neutralization tests against the attenuated strain XJCl3 were carried out and the antifusogenic activity of immunosera was studied by incubating serum with JV-infected Vero cells. Also the fusion activity in Vero cells infected with three JV strains was assayed. RESULTS AND CONCLUSIONS: A pathogenic strain XJ exhibited the highest fusogenic activity at pH 5. Syncytium formation was prevented by patients' sera obtained from different geographical locations, independently of time of infection. However, when Vero cells were infected with XJ, a significant reduction of syncytium production was observed, though the level of inhibition was lower than that detected in other JV strains-infected cells. These results could be explained by the existence of a conserved domain on JV proteins and also antigenic heterogeneity among strains.

Metabolism of platelet-activating factor in neutrophils isolated from Pichinde virus-infected guinea pigs.

Metabolism of platelet-activating factor (PAF) was studied in cultured polymorphonuclear neutrophils (PMNs) obtained from Pichinde virus-infected strain 13 guinea pigs. Neutrophils obtained from control and infected guinea pigs on postinoculation days 10 and 14 were incubated with [3H]lyso-PAF, [3H]PAF, or [3H]acetate. After incubation for 1 h at 37 degrees C, formation of [3H]acyl-PAF from either [3H]lyso-PAF or [3H]PAF increased significantly in PMNs from infected guinea pigs compared to control PMNs. Furthermore, total radioactivity from [3H]lyso-PAF or [3H]PAF was higher in PMNs from infected animals than in those from controls. However, compared to PAF production by control PMNs, production of PAF by infected PMNs was unchanged. These results suggest that PMNs may not be the major source of increased blood PAF levels during Pichinde viral disease.

Factor VIII: C and factor VIII R: Ag in Argentine hemorrhagic fever.

Factor VIII procoagulant activity (F VIII:C) and factor VIII related antigen (F VIII R: Ag) were investigated in 35 patients with Argentine hemorrhagic fever. Since the results obtained in the three clinical forms of the disease were not significantly different, they were tabulated altogether. F VIII:C was low in early stages of the disease but increased progressively in later days (days 5-6: 0.54 +/- 0.10 I. U/ml; days 13-14: 0.95 +/- 0.13 I.U./ml). In contrast, the levels of F VIII R: Ag were high all along the disease and they returned to normal values during the convalescence period (days 5-6; 2.58 +/- 0.54 I.U./ml; day 30: 1.30 +/- 0.14 I.U./ml). The levels of F VIII R: ag were similar in samples drawn before (11 cases) or after (10 cases) the treatment with immune plasma infusion. Plasma samples from 12 patients were studied by two-dimensional immunoelectrophoresis. The only abnormality found was increased height of the immune precipitation arc.

Early markers of blood coagulation and fibrinolysis activation in Argentine hemorrhagic fever.

Junin virus, an arenaviridae, is the etiological agent of Argentine hemorrhagic fever. In addition to thrombocytopenia, patients present several alterations in both the blood coagulation and the fibrinolytic system, but diffuse intravascular coagulation could not be demonstrated. To investigate further the activation status of the two systems, levels of thrombin-antithrombin complexes (TAT), prothrombin fragment 1 + 2, protein C, total and free protein S, C4bBP, antithrombin III, t-PA, PAI-1 and D-dimer were measured. Fourteen patients with a confirmed diagnosis of Argentine hemorrhagic fever were included in the study, 2 were severe, 3 moderate and 9 mild clinical cases, but hemorrhages were slight throughout. Blood samples were collected for 6 consecutive days on admission and on remission. At admission TAT and F1 + 2 levels were increased in 13/14 patients, reaching 0.33 nM (0.06-0.87) and 2.16 nM (0.96-6.5), respectively. PC was low in 4 cases, fPS in 6 and tPS in 2, whereas C4bBP and ATIII values were within normal range. t-PA and D-dimer levels were high in 11/14 patients, reaching 20 ng/ml (2.7-106) and 1660 ng/ml (877-3780) respectively, while PAI-1 was considerably increased in the 2 severe cases and normal in the remainder. These results suggest low level though persistent process of blood coagulation and fibrinolysis activation in this viral hemorrhagic disease. We believe these abnormalities may lead to the well described bleeding manifestations in these patients.

The African green monkey as an alternate primate host for studying Machupo virus infection.

African green monkeys (Cercopithecus aethiops) are highly susceptible to Bolivian hemorrhagic fever (BHF). Six monkeys were inoculated with 1,000 plague-forming units of Machupo virus, the etiologic agent of BHF. They were observed and monitored for clinical signs, body temperature, viremia, hematologic changes, and virus neutralizing antibody. Onset of fever, anorexia, and depression was noted on days 3 to 6 postinoculation. These and other signs increased in severity and all monkeys died: 5 of 6 died by day 13 and one survived until day 24. The median time to death for the group was 12.5 days. The mean value for hematocrit determinations gradually decreased to 30 on day 10 but subsequently increased. Mean neutrophil and lymphocyte values increased slightly until day 3, and then decreased to minimal values of 3,000 and 2,000, respectively, on day 10. Four monkeys were viremic by day 7 and all were viremic on day 10. The monkey that survived until day 24 had a neutralizing antibody titer of 1:32 on day 14 and appeared to recover from the initial acute illness by day 16. It died following onset of severe neurologic signs on day 23. BHF in the African green monkey is similar to the disease described in two species of macaques.

Studies of the coagulation system in arenaviral hemorrhagic fever: experimental infection of strain 13 guinea pigs with Pichinde virus.

Significant coagulation abnormalities were associated with experimental infection of strain 13 guinea pigs with Pichinde virus, an arenavirus related to the virulent human pathogens Junin, Machupo, and Lassa viruses. Infected animals developed decreased activity of multiple coagulation factors, decreased antithrombin III levels, high levels of fibrin-fibrinogen degradation products, impaired platelet function, and thrombocytopenia. Testing for the presence of a coagulation inhibitor revealed a pattern consistent with factor deficiency. Fibrin thrombi were not found at necropsy. The findings of high fibrin-fibrinogen degradation product levels and decreased antithrombin III levels, in association with decreased activity of multiple coagulation factors and thrombocytopenia, suggest that intravascular coagulation is a feature of this experimental infection. The demonstration of abnormal platelet function is also significant, as this could contribute to defective hemostasis despite the moderate thrombocytopenia which usually occurs in arenaviral disease.

Effect of persistent infection with Junin virus on growth and reproduction of its natural reservoir, Calomys musculinus.

The effect of infection with Junin virus on growth and reproduction of its natural reservoir, Calomys musculinus, was studied. Eighty-five C. musculinus were inoculated intranasally at birth with 100 TCID50 of Cba An 9446 strain of Junin virus and observed for 480 days. No clinical signs of neurologic illness were registered. Infected animals showed an increased mortality rate of up to 70% between days 24-40 post-infection. This period of high mortality was preceded by low weight gain during lactation and registered until 60 days. From day 14 post-infection until day 480, Junin virus was recovered from blood, urine, and oral swab in all animals checked at any time. By day 480 post-infection, 100% of survivors showed widespread viral dissemination in brain, spleen, kidneys, and salivary glands. There was marked reduction in reproductive efficiency among infected animals. Out of 15 mating pairs, 2 (13.3%) littered at least once compared to 60% in the control group. The reduction of fertility and the altered survival rate of Junin virus-infected C. musculinus indicate that vertical transmission mechanisms per se are insufficient to maintain the infection in successive generations in the absence of horizontal transmission.

A plasma inhibitor of platelet aggregation in patients with Argentine hemorrhagic fever.

Hemorrhage in patients with Lassa fever is associated with the presence of a circulating plasma inhibitor of platelet aggregation. This study was to determine whether patients with Argentine hemorrhagic fever (AHF) develop a similar inhibitor. Normal platelets showed significantly weaker aggregation responses to a sub-maximal dose of adenosine diphosphate (ADP) when mixed with plasma from 10 patients with AHF (mean percent of control +/- 1 SE = 57.2 +/- 6.7%) compared to those mixed with plasma from 9 viral control patients (79.5 +/- 4.1%; P less than 0.05) and 9 febrile patients with septicemia (103.8 +/- 3%; P less than 0.001). Plasma from 3 patients with severe AHF inhibited in a dose-dependent fashion the aggregation responses of normal platelets to collagen, sodium arachidonate, a calcium ionophore (A23187), and ristocetin; none of 4 samples from convalescent AHF patients showed this inhibitory activity. The platelet inhibition was sudden in onset and unaffected by a 30 min pre-incubation, not neutralized by convalescent plasma with high titer antibody to Junin virus, and abolished after heating plasma from an AHF patient at 56 degrees C for 30 min. Hemorrhage in AHF is associated with the presence of a circulating inhibitor of platelet aggregation, and disturbed hemostasis in arenavirus-induced hemorrhagic fevers may have a common basis.

Plasminogen abnormalities in patients with Argentine hemorrhagic fever.

Plasminogen, alpha 2-antiplasmin, alpha 2-macroglobulin, alpha 1-antitrypsin and fibrinogen degradation products (FDP) were studied in 45 patients with Argentine hemorrhagic fever. Patients were grouped into: 17 mild, 14 moderate and 14 severe cases. Plasminogen antigen level and functional activity were found to be reduced in the moderate and severe groups, when compared to the results obtained at recovery. The functional activity of alpha 2-antiplasmin was within the normal range, except for a slight decrease on days 10-11, alpha 2-macroglobulin remained normal during the course of illness. alpha 1-antitrypsin also remained normal except on days 10-11, when an increase in the antigen level was noted. FDP titre was normal (less than 10 micrograms/ml) in all patients during the course of disease. Plasminogen decrease was not attributable to liver insufficiency neither to a primary nor secondary fibrinolysis. The decreased antigen and reduced functionality of plasminogen in these patients we believe is related to proteolytic degradation by leukocyte enzymes.

Junin virus infection of Calithrix jacchus: haematological findings.

Haematological changes produced by experimental Junin virus infection of a platyrrhine monkey, Callithrix jacchus were studied. Normocytic and normochromic anaemia appeared after 7 days post infection (p. i.), and increased steadily until day 21 p. i. Reticulocytes and circulating erythroblasts were elevated during the anaemia, reached a peak on day 7 p. i., and disappeared later. Platelets and leukocytes showed a significant decrease from days 14 and 18 p. i., respectively. These alterations could be attributed to the damage of bone marrow and lymphatic tissue.

[Blood parameters variation in Calomys musculinus infected with Junin virus, strain XJCl3]. / Variación de parámetros sanguíneos en Calomys musculinus infectados con virus Junin, cepa XJCl3.

The aim of this study was to analyze the alterations in homeostasis induced by Junin virus during acute and persistent infection of C. musculinus. Virus presence in brain, hematological response and glycemia levels were evaluated. Newborn C. musculinus inoculated with 4000 DL50 of Junin virus, strain XJCl3 by intraperitoneal route developed a typical acute disease, with 50-70% mortality. Virus was isolated from brain starting day 6 post-infection (Fig. 1) and the peak titer (10(8) DL50/ml) was reached at 12 days post-infection. Neutralizing anti-Junin virus antibodies were detected from day 11 post-infection and all chronically infected animals developed persistent levels of neutralizing antibodies. In the acute stage of infection, 40% of the animals developed lymphopenia and neutrophilia (Fig. 2) while a slight variation was observed in the monocyte population. An important hypoglycemia was also seen in the acute infection (mean = 3.52 mmol/l) in comparison with control values (mean = 6.21 mmol/l), p less than 0.01 (Fig. 3). By contrast during the chronic stage of infection, neither hematological parameters (Table 2) varied between infected and control animals.

[Rapid serologic diagnosis of Argentinian hemorrhagic fever in whole blood]. / Diagnóstico serológico rápido de fiebre hemorrágica Argentina en sangre entera.

The usefulness of a method for detection of antibodies against Junin virus in whole blood was tested. N: NIH adult mice were inoculated with 10(3) PFU of attenuated XJ-Clon 3 Junin virus strain by intraperitoneal route and blood was obtained by retro-orbital puncture at 21 days post-infection. One blood aliquot (50 microliters) was collected in tubes containing a stabilizer solution for whole blood and another was processed for serum obtention. Immunofluorescent antibodies were tested on spot slides of a BHK/21 cell line persistently infected with Junin virus. High antibody titers (1/64 to 1/256) were detected in both whole blood and serum, with 66% coincidence between both procedures. These results show that the method of detection of antibodies in whole blood would be useful to test quickly for anti-Junin virus antibodies in seroepidemiologic studies, in endemic areas.

[Isolation of Junin virus from blood and peripheral lymphocytes of infected Calomys musculinus]. / Aislamiento de virus Junín a partir de sangre y linfo-monocitos periféricos de Calomys musculinus infectados.

Neonatal Calomys musculinus experimental infection with Junín virus (JV) XJCl3 strain causes either death or a persistent infection in the major part of surviving animals. JV can be isolated from peritoneal macrophages early during infection, and from brain and salivary glands during the chronic state of disease. It was of interest to investigate the appearance of virus in blood of infected animals. For this purpose, we decided to study the development of viremia in inoculated cricetids. A high frequency of viremia was registered during the acute state of disease (figure 1), which became sporadic approximately from 20 days post-infection onwards. Considering the results above mentioned, the characteristic lymphotropism of arenaviruses and the well-known JV replication in human mononuclear cells, cultures of lympho-monocytes obtained from blood of infected C. musculinus were done in order to investigate the eventual detection of infectious virus in the supernatants. JV was isolated, although in low titres, from cultures established with mononuclear cells belonging to animals in the acute state of disease (table 1); in the case of chronically infected cricetids, attempts to isolate JV were negative. These results show that viremia is currently detected in experimentally infected C. musculinus and that circulating mononuclear cells are permissive for JV multiplication, during the acute state of disease.

[Experimental infection of guinea pigs with Tacaribe virus: effect on the functioning of the immunocompetent system]. / Infección experimental del cobayo con virus Tacaribe: acci n sobre lafunción del aparato inmunocompetente.

Tacaribe virus is the member most closely related to Junín virus within the Tacaribe complex. It has been demonstrated that both viruses are indistinguishable by complement-fixation, due to the high cross-reactivity. However, adult guinea pigs are highly sensitive to infection with the XJ pathogenic strain of Junín virus whereas Tacaribe virus is nonpathogenic for this species. Furthermore this last virus protects them against Junín virus. The XJ strain reduces the immune response of guinea pigs to many antigens. Both the humoral response and the hypersensitivity of the Arthus type have been reduced in infected animals. Considering that Tacaribe virus could be used as vaccine antigen, the purpose of the present study was to investigate the effect of Tacaribe infection on the immune system of guinea pigs. The data reported here supports earlier findings showing that the XJ strain of Junín virus suppresses humoral immune response as indicated by lower precipitating antibody titers to ovoalbumin (which contributed to milder Arthus cutaneous reactivity) and a significant depression of plaque-forming cells to sheep erythrocytes. In contrast, Tacaribe-infected guinea pigs did not show detectable immunosuppression employing the same models. Similar results were found when the cell-mediated immunity was investigated. Tacaribe-infected guinea pigs had a normal immune response to contact sensitivity to 2-4 dinitro-1-fluorobenzene as demonstrated by measuring ear swelling and unmodified tuberculin reaction, after injection with complete Freund's adjuvant. Our results and those of previous investigations justify the consideration of Tacaribe immunization as an approach to the prophylaxis of Argentine Hemorrhagic Fever.