Safety and immunogenicity of a live-attenuated Junin (Argentine hemorrhagic fever) vaccine in rhesus macaques.

The safety and immunogenicity of Candid #1, a live-attenuated Junin virus vaccine, were evaluated in rhesus macaques. Candid #1 was inoculated subcutaneously in graded doses ranging from 16 to 127,200 plaque-forming units (PFU) into four groups of five animals each; four controls received saline. There was no significant effect of the immunization on any physical, hematologic, or biochemical parameter measured. Junin virus was recovered by cocultivation from peripheral blood mononuclear cells (PBMC) of 14 (70%) of 20 animals from 1 to 21 days after immunization; 27 (12%) of 223 PBMC samples that represented animals in all four dose groups were positive. In contrast, virus was recovered from the plasma of only two of 20 macaques (two of 225 samples [0.9%]), and only once (by amplification) from throat swabs. No evidence of reversion was detected in any blood isolate. All animals developed a detectable neutralizing antibody response following vaccination. These results indicate that Candid #1 is safe and immunogenic in nonhuman primates.

Candid No. 1 Argentine hemorrhagic fever vaccine protects against lethal Junin virus challenge in rhesus macaques.

The protective efficacy of Candid No. 1, a live-attenuated vaccine against Argentine hemorrhagic fever (AHF), was evaluated in non-human primates. Twenty rhesus macaques immunized 3 months previously with graded doses of Candid No. 1 (16-127, 000 PFU), as well as 4 placebo-inoculated controls, were challenged with 4.41 log10 PFU of virulent P3790 strain Junin virus. All controls developed severe clinical disease; 3 of 4 died. In contrast, all vaccinated animals were fully protected; none developed any signs of AHF during a 105-day follow-up period. Viremia and virus shedding were readily detected in all placebo-vaccinated controls, while virus could be recovered only once (by amplification) from throat swabs of 2 Candid No. 1 vaccinees on day 21. Vigorous secondary-type neutralizing and immunofluorescent antibody responses were seen in most vaccinees that had received 3 log10 PFU Candid No. 1 or fewer; all others, including those receiving 127,200 PFU, maintained relatively stable titers during follow-up. Candid No. 1 was highly immunogenic and fully protective against lethal Junin virus challenge in rhesus macaques, even at extremely low (16 PFU) vaccine doses.

A refined complement-enhanced neutralization test for detecting antibodies to Junin virus.

A refined, complement-enhanced, plaque-reduction neutralization test was developed for measuring neutralizing antibodies against Junin (Argentine hemorrhagic fever) virus. The assay measured neutralizing antibodies after natural as well as vaccine-induced Junin virus infections. Among vaccinated individuals, titers were 2-4-fold higher than those obtained with conventional assays, without loss of specificity. Enhanced sensitivity was achieved by using a standardized complement source (vs human or animal serum) for virus dilution, incubation of virus-serum mixtures at 36 degrees C for 2 h (vs overnight at 4 degrees C) prior to plaque assay, control of age and density of cell monolayers, and variation in overlay conditions.

Ribavirin prophylaxis and therapy for experimental argentine hemorrhagic fever.

Junin virus-infected rhesus macaques received prophylactic and therapeutic ribavirin to assess the potential of this drug for treating humans with Argentine hemorrhagic fever. When ribavirin was administered intramuscularly at the time of experimental infection with the lethal P3790 strain of Junin virus, all animals were protected from clinical disease. A delay in the initiation of therapy until after the onset of illness resulted in improvement and resolution of systemic signs of disease; however, survivors subsequently developed a late-onset central nervous system infection which was fatal in two of three animals. Side effects of ribavirin included thrombocytosis and severe anemia, both of which resolved promptly on withdrawal of drug therapy. Results of this study suggest that ribavirin may prove useful in treating humans with Argentine hemorrhagic fever.

Experimental Argentine hemorrhagic fever in rhesus macaques: virus-specific variations in pathology.

Two isolates of Junin virus (Espindola and Ledesma) inoculated into rhesus macaques produced distinct lesions which were strain-constant and similar to reported human cases of Argentine hemorrhagic fever. The Espindola isolate was associated with hemorrhagia, necrosis of bone marrow, and mild hepatocellular necrosis. Ledesma isolate was associated with pronounced polioencephalomyelitis and autonomic ganglioneuritis, but very mild or absent hepatocellular necrosis, bone marrow necrosis, and hemorrhagia. Deaths of Espindola-infected macaques were usually attributed to hemorrhagia with severe secondary bacterial infections, while in Ledesma-infected macaques, death was associated either with early severe secondary bacterial infections or slowly progressive polioencephalomyelitis. These two Junin virus isolates demonstrated hemorrhagic viscerotropism or neurotropism in macaques, suggesting that the variety of Argentine hemorrhagic fever syndromes in man may be virus-isolate determined.

Virus-specific factors in experimental Argentine hemorrhagic fever in rhesus macaques.

A nonhuman primate model for Argentine hemorrhagic fever has been developed that closely mimics the human clinical syndrome. Parenteral infection of adult Macaca mulatta with low-passage isolates of two Junin viral strains resulted in distinctive hemorrhagic or neurological disease in rhesus macaques that correlated with clinical illness patterns present in the humans from whom the viral strains were obtained. Transient leukopenia, together with thrombocytopenia and secondary bacterial septicemia, were documented among animals infected with both viral strains. In contrast, differing patterns of viremia, oropharyngeal viral shedding, and antibody response occurred in the two virus-infected groups. These results, together with postmortem virologic and histopathologic findings, suggest that viral-strain-specific factors are important determinants of clinical disease patterns in this model system.

Arenavirus concentration by molecular filtration.

Liter volumes of a human arenavirus pathogen (Machupo) and a nonpathogen (Tacaribe) were concentrated 30 to 100 times in less than 90 min without significant loss of particle infectivity.

Growth of human lymphoid cells (Raji strain) in a five-liter fermentor.

Procedures used to produce an established line of mammalian cells (L cell) in a New Brunswick fermentor were adapted to propagate human lymphoid cells in suspended culture. Control of pH within defined limits was more effective for regulation of cell metabolism than control of oxidation-reduction potential. An unusually high rate of agitation was required.

Ultrafiltration as a method for concentrating Rift Valley fever virus grown in tissue culture.

Filtration by means of a Diaflo ultrafilter was used to concentrate three 1,000-ml lots and one 3,000-ml lot of tissue culture-grown Rift Valley fever virus. Quantitation of both infectivity and total protein was achieved. Water treatment with continued ultrafiltration of the virus concentrate provided a final virus product approximately 99.25% free of low-molecular-weight materials originally present in the growth medium.

Growth of pathogenic virus in a large-scale tissue culture system.

A model system is described for the mass propagation of Rift Valley fever (RVF) virus, utilizing large-volume fermentor units for suspension culture of tissue cells and the subsequent production of virus. Comparisons between laboratory- and fermentor-scale operations of tissue cell growth gave equivalent results. Cell viability dropped 24 to 30 hr postinfection with a subsequent virus yield between 10(8.0) and 10(9.0) mouse intracerebral median lethal doses per milliliter. Infecting volumes of tissue cell culture (20- or 40-liter working volumes) had no apparent effect on virus yields. Tissue cells grown under either oxidation-reduction potential- and pH-controlled or uncontrolled conditions showed little or no difference in their ability to produce RVF virus. We believe this tissue cell virus process to have potential application for large-scale production of vaccines for human or veterinary use or for the mass propagation of certain carcinogenic viruses for cancer research, once use of established lines for this purpose is accepted.

Concentration of Rift Valley fever and Chikungunya viruses by precipitation.

Simple and efficient methods for concentrating Rift Valley fever (RVF) virus and chikungunya (CHIK) virus are described. Ammonium sulfate, potassium sulfate, or alcohol was used as a precipitating agent and the precipitate was resuspended to volumes suitable for further processing and purification. The methods permitted concentration of live RVF virus and CHIK virus about 100-fold with negligible losses of virus. RVF virus retained a high level of infectivity with potassium aluminum sulfate and alcohol, but CHIK virus retained a higher infectivity level with ammonium sulfate than with potassium aluminum sulfate. The data indicate that serum plays an important role in the concentration of both viruses, at least when the sulfate methods are used.

Interacting factors that influence long-term storage of live Pasteurella tularensis vaccine and Rift Valley fever virus.

Studies were conducted on the interaction of various parameters which affect the storage stability and growth potential of liquid cultures of Pasteurella tularensis live vaccine strain (LVS) and Rift Valley fever virus Van Wyk strain (RVFV). Storage variables studied with LVS included four storage temperatures (4, -20, -65, -175 C), single and multiple freeze-thaw cycles, two freezing and two thawing rates (slow and fast), various inoculum levels (1, 3, 5, and 10%) for the determination of growth potential, and the retention of immunizing potential (mice and guinea pig) after storage. Neither the freezing rate nor the number of freeze-thaw cycles seriously affected the growth of LVS after storage at -175C; however, the slow rate of thaw proved deleterious as were all temperatures of storage except -175 C after 1 year of storage, as shown by both criteria of evaluation. RVFV produced in two combinations of cell lines and media (LM cell line-199 peptone medium and LDR cell line-Eagle's minimum essential medium) was stored at three serum levels (10, 20, 40%), three pH values (6.2., 7.0, 7.8), and three temperatures (-20, -65, -175 C). These studies indicated: (i) virus produced in the LDR cell line and Eagle's medium was more stable than that produced in the LM cell line and 199 peptone medium for either short- or long-term storage; (ii) serum levels did not affect stability; and (iii) low pH resulted in losses during long-term storage under all conditions tested. Thus, cryogenic storage is advantageous for stock culture maintenance of bacteria and viruses and for other similar applications.

Purification and properties of in vitro-produced anthrax toxin components.

The three components of the toxin of Bacillus anthracis, edema factor (EF), protective antigen (PA), and lethal factor (LF), were purified 197-, 156-, and 1,025- fold, with 38, 78, and 11% recovery, respectively. Each purified component was serologically active, distinct, and free from the other components. The purified EF produced edema when mixed with PA, and the purified PA was an active immunogen. The components did not appear to be simple proteins by spectrophotometric analysis. As they were purified, the pH range in which they were most stable narrowed, centering between pH 7.4 and 7.8. Heat readily destroyed the biological activity of the components but not their serological activity. The rat lethality test showed that, with a constant amount of LF and an increasing amount of PA, the time to death reached a minimum and then was extended. When an increasing amount of LF was added to a constant amount of PA, the time to death became shorter as more LF was added. The biological, immunological, and serological properties of the components were shown to vary independently with storage and extent of purification so that serological activity was not always directly correlated with biological activity. Evidence is presented that the components can exist in different molecular configurations or as aggregates, and that this property is influenced by the state of component purity and by the environment.