Machupo Virus with Mutations in the Transmembrane Domain and Glycosylation Sites of the Glycoprotein Is Attenuated and Immunogenic in Animal Models of Bolivian Hemorrhagic Fever.

Several highly pathogenic mammarenaviruses cause severe hemorrhagic and neurologic disease in humans for which vaccines and antivirals are limited or unavailable. New World (NW) mammarenavirus Machupo virus (MACV) infection causes Bolivian hemorrhagic fever in humans. We previously reported that the disruption of specific N-linked glycan sites on the glycoprotein (GPC) partially attenuates MACV in an interferon alpha/beta and gamma (IFN-α/ß and -γ) receptor knockout (R-/-) mouse model. However, some capability to induce neurological pathology still remained. The highly pathogenic Junin virus (JUNV) is another NW arenavirus closely related to MACV. An F427I substitution in the GPC transmembrane domain (TMD) rendered JUNV attenuated in a lethal mouse model after intracranial inoculation. In this study, we rationally designed and rescued a MACV containing mutations at two glycosylation sites and the corresponding F438I substitution in the GPC TMD. The MACV mutant is fully attenuated in IFN-α/ß and -γ R-/- mice and outbred guinea pigs. Furthermore, inoculation with this mutant MACV completely protected guinea pigs from wild-type MACV lethal challenge. Last, we found the GPC TMD F438I substitution greatly impaired MACV growth in neuronal cell lines of mouse and human origins. Our results highlight the critical roles of the glycans and the TMD on the GPC in arenavirus virulence, which provide insight into the rational design of potential vaccine candidates for highly pathogenic arenaviruses. IMPORTANCE For arenaviruses, the only vaccine available is the live attenuated Candid#1 vaccine, a JUNV vaccine approved in Argentina. We and others have found that the glycans on GPC and the F427 residue in the GPC TMD are important for virulence of JUNV. Nevertheless, mutating either of them is not sufficient for full and stable attenuation of JUNV. Using reverse genetics, we disrupted specific glycosylation sites on MACV GPC and also introduced the corresponding F438I substitution in the GPC TMD. This MACV mutant is fully attenuated in two animal models and protects animals from lethal infection. Thus, our studies highlight the feasibility of rational attenuation of highly pathogenic arenaviruses for vaccine development. Another important finding from this study is that the F438I substitution in GPC TMD could substantially affect MACV replication in neurons. Future studies are warranted to elucidate the underlying mechanism and the implication of this mutation in arenavirus neural tropism.

Antibody-Based Inhibition of Pathogenic New World Hemorrhagic Fever Mammarenaviruses by Steric Occlusion of the Human Transferrin Receptor 1 Apical Domain.

Pathogenic clade B New World mammarenaviruses (NWM) can cause Argentine, Venezuelan, Brazilian, and Bolivian hemorrhagic fevers. Sequence variability among NWM glycoproteins (GP) poses a challenge to the development of broadly neutralizing therapeutics against the entire clade of viruses. However, blockade of their shared binding site on the apical domain of human transferrin receptor 1 (hTfR1/CD71) presents an opportunity for the development of effective and broadly neutralizing therapeutics. Here, we demonstrate that the murine monoclonal antibody OKT9, which targets the apical domain of hTfR1, can sterically block cellular entry by viral particles presenting clade B NWM glycoproteins (GP1-GP2). OKT9 blockade is also effective against viral particles pseudotyped with glycoproteins of a recently identified pathogenic Sabia-like virus. With nanomolar affinity for hTfR1, the OKT9 antigen binding fragment (OKT9-Fab) sterically blocks clade B NWM-GP1s and reduces infectivity of an attenuated strain of Junin virus. Binding of OKT9 to the hTfR1 ectodomain in its soluble, dimeric state produces stable assemblies that are observable by negative-stain electron microscopy. A model of the OKT9-sTfR1 complex, informed by the known crystallographic structure of sTfR1 and a newly determined structure of the OKT9 antigen binding fragment (Fab), suggests that OKT9 and the Machupo virus GP1 share a binding site on the hTfR1 apical domain. The structural basis for this interaction presents a framework for the design and development of high-affinity, broadly acting agents targeting clade B NWMs. IMPORTANCE Pathogenic clade B NWMs cause grave infectious diseases, the South American hemorrhagic fevers. Their etiological agents are Junin (JUNV), Guanarito (GTOV), Sabiá (SABV), Machupo (MACV), Chapare (CHAV), and a new Sabiá-like (SABV-L) virus recently identified in Brazil. These are priority A pathogens due to their high infectivity and mortality, their potential for person-to-person transmission, and the limited availability of effective therapeutics and vaccines to curb their effects. While low homology between surface glycoproteins of NWMs foils efforts to develop broadly neutralizing therapies targeting NWMs, this work provides structural evidence that OKT9, a monoclonal antibody targeting a single NWM glycoprotein binding site on hTfR1, can efficiently prevent their entry into cells.

Junin Virus Triggers Macrophage Activation and Modulates Polarization According to Viral Strain Pathogenicity.

The New World arenavirus Junin (JUNV) is the etiological agent of Argentine hemorrhagic fever (AHF). Previous studies of human macrophage infection by the Old-World arenaviruses Mopeia and Lassa showed that while the non-pathogenic Mopeia virus replicates and activates human macrophages, the pathogenic Lassa virus replicates but fails to activate human macrophages. Less is known in regard to the impact of New World arenavirus infection on the human macrophage immune response. Macrophage activation is critical for controlling infections but could also be usurped favoring immune evasion. Therefore, it is crucial to understand how the JUNV infection modulates macrophage plasticity to clarify its role in AHF pathogenesis. With this aim in mind, we compared infection with the attenuated Candid 1 (C#1) or the pathogenic P strains of the JUNV virus in human macrophage cultures. The results showed that both JUNV strains similarly replicated and induced morphological changes as early as 1 day post-infection. However, both strains differentially induced the expression of CD71, the receptor for cell entry, the activation and maturation molecules CD80, CD86, and HLA-DR and selectively modulated cytokine production. Higher levels of TNF-α, IL-10, and IL-12 were detected with C#1 strain, while the P strain induced only higher levels of IL-6. We also found that C#1 strain infection skewed macrophage polarization to M1, whereas the P strain shifted the response to an M2 phenotype. Interestingly, the MERTK receptor, that negatively regulates the immune response, was down-regulated by C#1 strain and up-regulated by P strain infection. Similarly, the target genes of MERTK activation, the cytokine suppressors SOCS1 and SOCS3, were also increased after P strain infection, in addition to IRF-1, that regulates type I IFN levels, which were higher with C#1 compared with P strain infection. Together, this differential activation/polarization pattern of macrophages elicited by P strain suggests a more evasive immune response and may have important implications in the pathogenesis of AHF and underpinning the development of new potential therapeutic strategies.

Protocol for the Production of a Vaccine Against Argentinian Hemorrhagic Fever.

Argentinian hemorrhagic Fever (AHF) is a febrile, acute disease caused by Junín virus (JUNV), a member of the Arenaviridae. Different approaches to obtain an effective antigen to prevent AHF using complete live or inactivated virus, as well as molecular constructs, have reached diverse development stages. This chapter refers to JUNV live attenuated vaccine strain Candid #1, currently used in Argentina to prevent AHF. A general standardized protocol used at Instituto Nacional de Enfermedades Virales Humanas (Pergamino, Pcia. Buenos Aires, Argentina) to manufacture the tissue culture derived Candid #1 vaccine is described. Intermediate stages like viral seeds and cell culture bank management, bulk vaccine manufacture, and finished product processing are also separately presented in terms of Production and Quality Control/Quality Assurance requirements, under the Adminitracion Nacional de Medicamentos, Alimentos y Tecnología Medica (ANMAT), the Argentine national regulatory authority.

Convergent immunological solutions to Argentine hemorrhagic fever virus neutralization.

Transmission of hemorrhagic fever New World arenaviruses from their rodent reservoirs to human populations poses substantial public health and economic dangers. These zoonotic events are enabled by the specific interaction between the New World arenaviral attachment glycoprotein, GP1, and cell surface human transferrin receptor (hTfR1). Here, we present the structural basis for how a mouse-derived neutralizing antibody (nAb), OD01, disrupts this interaction by targeting the receptor-binding surface of the GP1 glycoprotein from Junín virus (JUNV), a hemorrhagic fever arenavirus endemic in central Argentina. Comparison of our structure with that of a previously reported nAb complex (JUNV GP1-GD01) reveals largely overlapping epitopes but highly distinct antibody-binding modes. Despite differences in GP1 recognition, we find that both antibodies present a key tyrosine residue, albeit on different chains, that inserts into a central pocket on JUNV GP1 and effectively mimics the contacts made by the host TfR1. These data provide a molecular-level description of how antibodies derived from different germline origins arrive at equivalent immunological solutions to virus neutralization.

Absence of an N-Linked Glycosylation Motif in the Glycoprotein of the Live-Attenuated Argentine Hemorrhagic Fever Vaccine, Candid #1, Results in Its Improper Processing, and Reduced Surface Expression.

Junin virus (JUNV), a highly pathogenic New World arenavirus, is the causative agent of Argentine hemorrhagic fever (AHF). The live-attenuated Candid #1 (Can) strain currently serves as a vaccine for at-risk populations. We have previously shown that the Can glycoprotein (GPC) gene is the primary gene responsible for attenuation in a guinea pig model of AHF. However, the mechanisms through which the GPC contributes to the attenuation of the Can strain remain unknown. A more complete understanding of the mechanisms underlying the attenuation and immunogenicity of the Can strain will potentially allow for the rational design of additional safe and novel vaccines. Here, we provide a detailed comparison of both RNA and protein expression profiles between both inter- and intra-segment chimeric JUNV recombinant clones expressing combinations of genes from the Can strain and the pathogenic Romero (Rom) strain. The recombinant viruses that express Can GPC, which were shown to be attenuated in guinea pigs, displayed different RNA levels and GPC processing patterns as determined by Northern and Western blot analyses, respectively. Analysis of recombinant viruses containing amino acid substitutions selected at different mouse brain passages during the generation of Can revealed that altered Can GPC processing was primarily due to the T168A substitution within G1, which eliminates an N-linked glycosylation motif. Incorporation of the T168A substitution in the Rom GPC resulted in a Can-like processing pattern of Rom GPC. In addition, JUNV GPCs containing T168A substitution were retained within the endoplasmic reticulum (ER) and displayed significantly lower cell surface expression than wild-type Rom GPC. Interestingly, the reversion A168T in Can GPC significantly increased GPC expression at the cell surface. Our results demonstrate that recombinant JUNV (rJUNV) expressing Can GPC display markedly different protein expression and elevated genomic RNA expression when compared to viruses expressing Rom GPC. Additionally, our findings indicate that the N-linked glycosylation motif at amino acid positions 166-168 is important for trafficking of JUNV GPC to the cell surface, and the elimination of this motif interferes with the GPC release from the ER.

[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.

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

La Fiebre Hemorrágica Argentina es una enfermedad producida por el virus Junín. Para la prevención de esta enfermedad se obtuvo una vacuna efectiva denominada Candid#1. Durante un ensayo clínico realizado en el INEVH, dos cepas de virus Junín fueron aisladas de sangre periférica de dos voluntarios mediante co-cultivo de células mononucleares. El objetivo de este trabajo fue comparar las características fenotípicas de atenuación de esas dos cepas recuperadas de humanos con las de la vacuna Candid#1 utilizando los indicadores de atenuación desarrollados por Contigiani y Sabattini en 1977. A tal fin se midieron los índices de letalidad, infección y protección en cobayos y ratones de diferentes edades. Las tres cepas investigadas resultaron letales para ratones recién nacidos pero no para ratones de 10 a 12 días, ratones adultos ni cobayos, aun a la más baja dilución inoculada. Los cobayos inoculados con las cepas recuperadas de humanos y con la cepa Candid#1 no presentaron síntomas de enfermedad y mostraron estar protegidos cuando fueron desafiados con una cepa patógena. Los índices de infección y de protección hallados indican que estas cepas poseen elevada capacidad infectante y protectora en las especies animales aquí estudiadas. Estos resultados demuestran que las cepas de virus Junín aisladas de voluntarios inmunizados con Candid#1 mantienen el mismo fenotipo atenuado de la vacuna Candid#1 después de un pasaje por humanos.

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.

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

La Fiebre Hemorrágica Argentina es una enfermedad producida por el virus Junín. Para la prevención de esta enfermedad se obtuvo una vacuna efectiva denominada Candid#1. Durante un ensayo clínico realizado en el INEVH, dos cepas de virus Junín fueron aisladas de sangre periférica de dos voluntarios mediante co-cultivo de células mononucleares. El objetivo de este trabajo fue comparar las características fenotípicas de atenuación de esas dos cepas recuperadas de humanos con las de la vacuna Candid#1 utilizando los indicadores de atenuación desarrollados por Contigiani y Sabattini en 1977. A tal fin se midieron los índices de letalidad, infección y protección en cobayos y ratones de diferentes edades. Las tres cepas investigadas resultaron letales para ratones recién nacidos pero no para ratones de 10 a 12 días, ratones adultos ni cobayos, aun a la más baja dilución inoculada. Los cobayos inoculados con las cepas recuperadas de humanos y con la cepa Candid#1 no presentaron síntomas de enfermedad y mostraron estar protegidos cuando fueron desafiados con una cepa patógena. Los índices de infección y de protección hallados indican que estas cepas poseen elevada capacidad infectante y protectora en las especies animales aquí estudiadas. Estos resultados demuestran que las cepas de virus Junín aisladas de voluntarios inmunizados con Candid#1 mantienen el mismo fenotipo atenuado de la vacuna Candid#1 después de un pasaje por humanos.(AU)

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.(AU)

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

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.

Rescue from cloned cDNAs and in vivo characterization of recombinant pathogenic Romero and live-attenuated Candid #1 strains of Junin virus, the causative agent of Argentine hemorrhagic fever disease.

The New World arenavirus Junin virus (JUNV) is the causative agent of Argentine hemorrhagic fever (AHF), which is associated with high morbidity and significant mortality. Several pathogenic strains of JUNV have been documented, and a highly attenuated vaccine strain (Candid #1) was generated and used to vaccinate the human population at risk. The identification and functional characterization of viral genetic determinants associated with AHF and Candid #1 attenuation would contribute to the elucidation of the mechanisms contributing to AHF and the development of better vaccines and therapeutics. To this end, we used reverse genetics to rescue the pathogenic Romero and the attenuated Candid #1 strains of JUNV from cloned cDNAs. Both recombinant Candid #1 (rCandid #1) and Romero (rRomero) had the same growth properties and phenotypic features in cultured cells and in vivo as their corresponding parental viruses. Infection with rRomero caused 100% lethality in guinea pigs, whereas rCandid #1 infection was asymptomatic and provided protection against a lethal challenge with Romero. Notably, Romero and Candid #1 trans-acting proteins, L and NP, required for virus RNA replication and gene expression were exchangeable in a minigenome rescue assay. These findings support the feasibility of studies aimed at determining the contribution of each viral gene to JUNV pathogenesis and attenuation. In addition, we rescued Candid #1 viruses with three segments that efficiently expressed foreign genes introduced into their genomes. This finding opens the way for the development of a safe multivalent arenavirus vaccine.

[Candid#1 vaccine against Argentine hemorrhagic fever produced in Argentina. Immunogenicity and safety]. / Vacuna contra la fiebre hemorrágica argentina Candid#1 producida en la Argentina. Inmunogenicidad y seguridad.

A clinical study in 946 human volunteers was done to compare Candid #1 vaccine manufactured in Argentina with the vaccine produced in USA that had been previously used. The efficacy was evaluated using immunogenicity measured by the detection of neutralizing antibodies as a subrogate marker. Safety was evaluated comparing the rate of adverse events. Both vaccines showed a comparable rate of seroconversion, slightly higher than the efficacy estimated from previous studies (95.5%). There were no severe adverse events related to the vaccines. The general events considered related to the vaccines were not clinically relevant and disappeared either spontaneously or with symptomatic treatment. Similar rates of adverse events (29.9% for the Argentine vaccine and 35.0% for the USA vaccine) were found for both vaccines. These included: headache, weakness, myalgias, mild low blood cell (< 4,000/mm(3)) and platelet (< 150,000/mm(3)) counts, nausea and/or vomiting, fever, retroocular pain, dizziness, microhematuria, low backache and exantema. These results indicate that the vaccine Candid#1 manufactured in Argentina is equivalent to the manufactured in USA. These results allowed the National Institute of Human Viral Diseases (INEVH) to register the vaccine produced locally under the National Regulatory Authority (ANMAT).

Vacuna contra la fiebre hemorrágica argentina Candid#1 producida en la Argentina: Inmunogenicidad y seguridad / Candid#1 vaccine against Argentine Hemorrhagic Fever produced in Argentina: Immunogenicity and safety

Se realizó un estudio clínico en 946 voluntarios humanos sanos, donde se comparó la vacuna Candid#1 producida en Argentina con la elaborada en EE.UU., que había sido utilizada en estudios previos. Como objetivo primario se evaluó la equivalencia en la eficacia utilizando como marcador subrogante a la inmunogenicidad medida por detección de anticuerpos neutralizantes. Como objetivo secundario se evaluó la equivalencia en inocuidad comparando las tasas de reacciones adversas. Ambas vacunas mostraron una tasa equivalente de inmunogenicidad ligeramente superior al 95.5%, que es la eficacia estimada para Candid #1 en estudios previos. No se observaron eventos adversos graves relacionados con la vacuna. Los eventos adversos generales considerados relacionados fueron de escasa significación clínica y de resolución espontánea o con tratamiento sintomático; se presentaron en los receptores de ambas vacunas en tasas equivalentes (29.9% para la vacuna fabricada en la Argentina y 35.0% para la fabricada en EE.UU.), e incluyeron: cefalea, decaimiento, mialgias, plaquetopenia leve (< 150 000 plaquetas/mm³), náuseas y/o vómitos, leucopenia leve (< 4 000 blancos/mm³), fiebre, dolor retroocular, mareos, microhematuria, lumbalgia y exantema. Estos resultados indican que la vacuna Candid #1 elaborada en la Argentina es equivalente a la elaborada en los EE.UU. Este estudio permitió el registro del biológico producido en la Argentina ante la autoridad regulatoria del país (ANMAT).

A clinical study in 946 human volunteers was done to compare Candid #1 vaccine manufactured in Argentina with the vaccine produced in USA that had been previously used. The efficacy was evaluated using immunogenicity measured by the detection of neutralizing antibodies as a subrogate marker. Safety was evaluated comparing the rate of adverse events. Both vaccines showed a comparable rate of seroconversion, slighty higher than the efficacy estimated from previous studies (95.5%). There were no severe adverse events related to the vaccines. The general events considered related to the vaccines were not clinically relevant and disappeared either spontaneously or with symptomatic treatment. Similar rates of adverse events (29.9% for the Argentine vaccine and 35.0% for the USA vaccine) were found for both vaccines. These included: headache, weakness, myalgias, mild low blood cell (< 4 000/mm³) and platelet (< 150 000/mm³) counts, nausea and/or vomiting, fever, retroocular pain, dizziness, microhematuria, low backache and exantema. These results indicate that the vaccine Candid#1 manufactured in Argentina is equivalent to the manufactured in USA. These results allowed the National Institute of Human Viral Diseases (INEVH) to register the vaccine produced locally under the National Regulatory Authority (ANMAT).

Treatment of Argentine hemorrhagic fever.

Argentine hemorrhagic fever (AHF) is a rodent-borne illness caused by the arenavirus Junin that is endemic to the humid pampas of Argentina. AHF has had significant morbidity since its emergence in the 1950s, with a case-fatality rate of the illness without treatment between 15% and 30%. The use of a live attenuated vaccine has markedly reduced the incidence of AHF. Present specific therapy involves the transfusion of immune plasma in defined doses of neutralizing antibodies during the prodromal phase of illness. However, alternative forms of treatment are called for due to current difficulties in early detection of AHF, related to its decrease in incidence, troubles in maintaining adequate stocks of immune plasma, and the absence of effective therapies for severely ill patients that progress to a neurologic-hemorrhagic phase. Ribavirin might be a substitute for immune plasma, provided that the supply is guaranteed. Immune immunoglobulin or monoclonal antibodies should also be considered. New therapeutic options such as those being developed for systemic inflammatory syndromes should also be valuated in severe forms of AHF.

Immune response to vaccination against Argentine hemorrhagic Fever in an area where different arenaviruses coexist.

Neutralizing antibody (NT Ab) titers to Candid #1 (C#1) vaccine against Argentine hemorrhagic fever were studied for 2 years post-vaccination in 330 volunteers, to assess whether the kinetics and/or magnitude of this immune response is modified by previous infection with the arena viruses Junin (JUN) and lymphocytic choriomeningitis (LCM). A total of 160 volunteers received C#1, distributed as follows: without detectable pre-infection with arenaviruses (n = 54); with pre-existing antibodies to JUN (n = 55); with pre-existing antibodies to LCM (n = 51). The remaining 170 individuals received placebo. Levels of anti-JUN NT Ab displayed a trend in which titers increased with the virulence of the infecting strain, from C#1 (X = 49), through subclinical JUN infection (X = 229), vaccination following subclinical infection (X = 367) to JUN clinical infection (X =773). It was also found that the mean titer of NT Ab to C#1 did not vary significantly during 2 years of study and was: a) significantly lower than that elicited by wild strains of JUN, both clinical and subclinical infections (p < 0.01); b) significantly increased the titers of pre-existing anti-JUN Ab (p < 0.01); and c) was not modified by pre-existing anti-LCM Ab (p > 0.05). These data indicate that the immune response to C#1 boosts pre-existing immunity to JUN virus and is not changed by previous experience with LCM virus.

[Preclinical assay of candid #1 vaccine against Argentine Hemorrhagic Fever made in Argentina]. / Ensayo preclínico de la vacuna Candid #1 producida en Argentina contra la Fiebre Hemorrágica Argentina.

Candid #1 vaccine against Argentine Hemorrhagic Fever produced in USA versus lots of the same vaccine made in Argentina were compared in guinea pigs regarding safety, immunogenicity and protective efficacy against a challenge with pathogenic Junin virus. Lots No Exp 3, 7A and 8A of Argentine origin as well as lot TSI 5-1-92 from USA were inoculated in guinea pigs of 250-400 g in two consecutive assays. Ten animals inoculated with saline performed as normal controls in each experiment. Parameters studied were: a) temperature; b) body weight; c) neutralizing antibodies to Junin virus; d) response to viral challenge. Animals gained weight and remained normothermic up to the challenge. Guinea pigs that received Candid #1 from any manufacturer elicited neutralizing antibodies to Junin virus (titles from 40 to 81920) and survived to challenge whilst 8/10 animals died in each control group. Data presented demonstrated that Candid #1 vaccines from USA or Argentine manufacturers were equally safe, immunogenic and protective in guinea pigs.

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.

Ensayo preclinico de la vacuna candid #1 producida en Argentina contra la fiebre hemorragica Argentina / Preclinical assay of candid #1 vaccine against argentine hemorrhagic fever made in Argentina

Se comparó en cobayos la seguridad, inmunogenicidad y eficácia protectora de um lote de vacuna Candid #1 (C#1) fabricada en Estados Unidos de América (EE.UU.) y distintos lotes de la misma vacuna fabricados en Argentina (Arg.). El lote TSI 5-1-92 (EE.UU) y los lotes Exp N3, 7A y 8A (Arg) fueron inoculados (0.5ml, IM) en cobayos de 250400g. Para cada ensayo diez animales recibieron solución fisiológica y sirvieron como control. Todos fueron desafiados con la cepa patógena P23790 de vírus Junin. Se registro: a) temperatura rectal, b) peso corporal , c) presencia de anticuerpos neutralizantes (AcNT) pré y post-vacunación, d) respuesta al desafio . Todos los animales vacunados desarrollaron AcNT anti vírus Junin (rango= 4081920 y sobrevivieron al desafio. En cada grupo control 810 animales murieron (dia 23.3+_ 5.4 post- desaportada y los diferentes lotes de C#1 producidos en Argentina.

Ensayo preclinico de la vacuna candid #1 producida en Argentina contra la fiebre hemorragica Argentina / Preclinical assay of candid #1 vaccine against argentine hemorrhagic fever made in Argentina

Se comparó en cobayos la seguridad, inmunogenicidad y eficácia protectora de um lote de vacuna Candid #1 (C#1) fabricada en Estados Unidos de América (EE.UU.) y distintos lotes de la misma vacuna fabricados en Argentina (Arg.). El lote TSI 5-1-92 (EE.UU) y los lotes Exp N3, 7A y 8A (Arg) fueron inoculados (0.5ml, IM) en cobayos de 250400g. Para cada ensayo diez animales recibieron solución fisiológica y sirvieron como control. Todos fueron desafiados con la cepa patógena P23790 de vírus Junin. Se registro: a) temperatura rectal, b) peso corporal , c) presencia de anticuerpos neutralizantes (AcNT) pré y post-vacunación, d) respuesta al desafio . Todos los animales vacunados desarrollaron AcNT anti vírus Junin (rango= 4081920 y sobrevivieron al desafio. En cada grupo control 810 animales murieron (dia 23.3+_ 5.4 post- desaportada y los diferentes lotes de C#1 producidos en Argentina. (AU)