Lassa fever - full recovery without ribavarin treatment: a case report

Background

Lassa fever is a rodent-borne zoonosis that clinically manifests as an acute hemorrhagic fever. It is treated using ribavarin. Surviving Lassa fever without receiving the antiviral drug ribavarin is rare. Only few cases have been documented to date.

Case Presentation

We report a case of a 59-year old female with fever who was initially thought to have acute pyelonephritis and sepsis syndrome with background malaria. Further changes in her clinical state and laboratory tests led to a suspicion of Lassa fever. However at the time her laboratory confirmatory test for Lassa fever returned, her clinical state had improved and she made full recovery without receiving ribavarin. Her close contacts showed no evidence of Lassa virus infection.

Conclusion

This report adds to the literature on the natural history of Lassa fever; and that individuals may survive Lassa fever with conservative management of symptoms of the disease and its complications.     

Evaluating the immunogenicity and protective efficacy of a DNA vaccine encoding Lassa virus nucleoprotein

Several viruses in the Arenavirus genus of the family Arenaviridae cause severe, often fatal, hemorrhagic fever. One such virus, Lassa virus (LV), is a frequent cause of disease in Africa, and survivors often are left with substantial neurological impairment. The feasibility of protective immunization against LV infection, and the associated disease, has been demonstrated in animal models, using recombinant vaccinia viruses to deliver Lassa proteins. Circumstantial evidence implicates cellular immunity in this Lassa-induced protection, but this has not been confirmed. Here, we describe DNA vaccines that encode LV proteins. A single inoculation of a plasmid encoding full-length Lassa nucleoprotein (LNP) can induce CD8(+) T cell responses in mice and can protect against challenge with two arenaviruses, lymphocytic choriomeningitis virus (LCMV) and Pichinde virus (PV). A DNA minigene vaccine encoding a 9 amino acid sequence from LNP also induces CD8(+) T cells and protects against arenavirus challenge, thus confirming prior speculation that protective cellular immunity is induced by LV proteins.     

NK cells are strongly activated by Lassa and Mopeia virus-infected human macrophages in vitro but do not mediate virus suppression

Lassa virus (LASV) and Mopeia virus (MOPV) are closely related Arenaviruses. LASV causes hemorrhagic fever, whereas MOPV is not pathogenic. Both viruses display tropism for APCs such as DCs and macrophages. During viral infections, NK cells are involved in the clearance of infected cells and promote optimal immune responses by interacting with APCs. We used an in vitro model of human NK and APC coculture to study the role of NK cells and to characterize their interactions with APCs during LASV and MOPV infections. As expected, NK cells alone were neither infected nor activated by LASV and MOPV, and infected DCs did not activate NK cells. By contrast, LASV- and MOPV-infected macrophages activated NK cells, as shown by the upregulation of CD69, NKp30, and NKp44, the downregulation of CXCR3, and an increase in NK-cell proliferation. NK cells acquired enhanced cytotoxicity, as illustrated by the increase in granzyme B (GrzB) expression and killing of K562 targets, but did not produce IFN-γ. Contact between NK cells and infected macrophages and type I IFNs were essential for activation; however, NK cells could not kill infected cells and control infection. Overall, these findings show that MOPV- as well as pathogenic LASV-infected macrophages mediate NK-cell activation.     

Pathogenesis and immune response of vaccinated and unvaccinated rhesus monkeys to tick-borne encephalitis virus.

The rhesus monkey was used as a model for diseases caused by viruses of the tick-borne encephalitis virus complex to study the efficacy and safety of a commercial killed vaccine. Animals infected intravenously developed a subclinical infection with no histopathological lesions but with transient clinical chemical changes that included elevated transaminase, dehydrogenase, and creatine kinase activities and that declined as an immune response developed. The immune response was detected as neutralizing antibody in serum and serum antibody to several viral proteins. Antibodies to viral envelope protein and two other infected cell-specific polypeptides were also detected. Intranasal infection resulted in a disease resembling that in humans, except that no pyrexia was observed. Clinical chemical changes similar to those in intravenously infected monkeys developed, but most animals died before an immune response was mounted. Using this model, we have demonstrated that a commercial vaccine protects animals against a wild-type virus isolate and that it elicits an effective immune reaction without any evidence of an immune enhancement phenomenon or adverse side effects as judged by clinical observation, clinical chemistry, and histopathology.     

Molecular characterization of a reassortant virus derived from Lassa and Mopeia viruses

In this article we describe two new complete genomic sequences of Old World Arenaviruses: the Mopeia (MOP) virus and the reassortant MOP/LAS virus, clone 29, or ML29. This reassortant has the large (L) RNA from MOP virus and the small (S) RNA from Lassa (LAS) virus, Josiah strain. Recent studies showed that the ML29 virus is not pathogenic for mice, guinea pigs, or macaques, can completely protect guinea pigs from Lassa virus, and elicit vigorous cell-mediated immunity in immunized monkeys (Lukashevich, I. S., Patterson, J., Carrion, R., Moshkoff, D., Ticer, A., Zapata, J., Brasky, K., Geiger, R., Hubbard, G. B., Bryant, J., and Salvato, M. S., J Virol 79, 13934–13942, 2005). This is a molecular characterization of a reassortant virus, which has been put forward as a live attenuated vaccine candidate against Lassa Fever. Sequence analysis of this reassortant virus revealed 5 non-conservative amino acid substitutions that distinguished it from the parental LAS and MOP viruses. Three substitutions were found outside the conserved RNA-dependent RNA polymerase (RdRp) motifs. A fourth substitution was located between the glycoprotein (GPC)-cleavage site and the putative fusion peptide of GP2. The nucleocapsid protein (NP) contained a fifth substitution in the carboxyl-terminal region of the protein. Two mutations were found within each non-coding terminus of the L segment and one mutation was located in the 3′ non-coding region of the S segment of the MOP/LAS virus. ML29 mutations in its genomic termini may have implications for the genetic stability and replication efficiency of ML29 reassortant. The nucleotide sequence data for the of the S and L RNAs of the ML29 and Mopeia virus strains reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers: NC_006572-75.     

A hemagglutinin-based multipeptide construct elicits enhanced protective immune response in mice against influenza A virus infection

Multipeptide constructs, comprising adjacent sequences of the 317–341 intersubunit region of immature influenza A hemagglutinin (H1N1), were designed and the functional properties of these branched peptides were compared to that of the corresponding linear peptides. In vivo studies revealed that the immunogenicity of the peptides was dependent on the presence of the hydrophobic fusion peptide (comprised in FP3), encompassing the N-terminal 1–13 sequence of the HA2 subunit. Antibody and T cell recognition, however, was directed against the 317–329 HA1 sequence, comprised in the P4 peptide. Multiple copies of P4, covalently linked by branched lysine residues, significantly enhanced the efficiency of antibody binding and the capacity of peptides to elicit B- and T-cell responses. A fraction of peptide induced antibodies reacted with immature or with proteolitically cleaved hemagglutinin (HA) molecules pretreated at low pH. Immunization with a multipeptide construct, (P4)₄–FP3, not only resulted in elevated antibody and T cell responses but conferred enhanced protection against lethal A/PR/8/34 (H1N1) infection as compared to its subunit peptides. The beneficial functional properties of this artificial peptide antigen may be acquired by multiple properties including: (i) stabilized peptide conformation which promotes strong, polyvalent binding to both antibodies and MHC class II molecules; (ii) appropriate P4 conformation for antibody recognition stabilized by the covalently coupled fusion peptide, resulting in the production of virus cross reactive antibodies which inhibit the fusion activity of the virus; (iii) activation of peptide specific B cells which potentiate antigen presentation and peptide specific T cell responses; and (iv) generation of helper T cells which secrete lymphokines active in the resolution of infection.     

Procainamide elicits a selective autoantibody immune response.

The specificity of the in vivo humoral immune response elicited by procainamide was examined by solid-phase assays, immunofluorescence, immunoprecipitation and a cytotoxicity assay. Serial samples obtained from patients during their procainamide therapy showed a progressive increase in antibodies to histones and denatured DNA, and both activities decreased after discontinuation of therapy. In contrast antibodies to tetanus, human IgG (rheumatoid factor) and heterologous lymphocytes were unaffected by procainamide treatment, indicating that they were not drug-induced. Of 29 sera examined by protein-A-facilitated immunoprecipitation, four sera had antibody to ribosomal RNA and three sera immunoprecipitated a 40kD protein. Antinuclear antibodies were invariably present but absorption studies showed that these activities were due to anti-histone antibodies. These results indicate that procainamide-induced autoimmunity is characterized predominantly by an anti-histone and anti-denatured DNA immune response.     

Decoding arenavirus pathogenesis: essential roles for alpha-dystroglycan-virus interactions and the immune response

Pathogenesis following a virus infection results from interactions between the virus and its host. The outcome is determined by tipping the balance between virulence of the virus or susceptibility/resistance of the host to favor one or the other. This review focuses on two important members of the Old World arenavirus family: Lassa fever virus (LFV), a robust human pathogen that causes a severe acute hemorrhagic disease; and lymphocytic choriomeningitis virus (LCMV), also a human pathogen but better known in the context of its rodent model. Research with this model has uncovered and illuminated many of our current concepts in immunobiology and viral pathogenesis. Presented here are recent advances that form the framework for a better understanding of how viruses induce and maintain persistent infection as well as for the pathogenesis associated with acute LFV infection. A major component for understanding the pathogenesis of these arenaviruses revolves around study of the interaction of virus with its receptor, alpha-dystroglycan (α-DG).     

Neurovirulence of yellow fever 17DD vaccine virus to rhesus monkeys

The yellow fever 17D virus is attenuated and used for human vaccination. Two of its substrains, 17D-204 and 17DD, are used for vaccine production. One of the remarkable properties of this vaccine is limited viral replication in the host but with significant dissemination of the viral mass, yielding a robust and long-lived neutralizing antibody response. The vaccine has excellent records of efficacy and safety and is cheap, used as a single dose, and there are well-established production methodology and quality control procedures which include the monkey neurovirulence test (MNTV). The present study aims at a better understanding of YF 17DD virus attenuation and immunogenicity in the MNVT with special emphasis on viremia, seroconversion, clinical and histological lesions scores, and their intrinsic variability across the tests. Several MNVTs were performed using the secondary seed lot virus 17DD 102/84 totaling 49 rhesus monkeys. Viremia was never higher than the accepted limits established in international requirements, and high levels of neutralizing antibodies were observed in all animals. None of the animals showed visceral lesions. We found that the clinical scores for the same virus varied widely across the tests. There was a direct correlation between the clinical scores in animals with clinical signs of encephalitis and a higher degree of central nervous system (CNS) histological lesions, with an increase of lesions in areas of the CNS such as the substantia nigra, nucleus caudatus, intumescentia cervicalis, and intumescentia ventralis. The histological scores were shown to be less prone to individual variations and had a more homogeneous value distribution among the tests. Since 17DD 102/84 seed virus has been used for human vaccine production and immunization for 16 years with the vaccine being safe and efficacious, it demonstrates that the observed variations across the MNVTs do not influence its effect on humans.     

Cell-mediated immune response to respiratory syncytial virus infection in owl monkeys.

The suitability of owl monkeys as experimental models to study the cellular immune response to respiratory syncytial virus (RSV) infection was examined. Seronegative owl monkeys inoculated intranasally with RSV shed large quantities of virus and developed clinically evident upper respiratory disease. RSV infected monkeys had significant lymphoproliferative responses to RSV antigen by 4 weeks post-infection. In contrast, no positive blastogenic responses were elicited during the acute phase of illness. An in vitro 51Cr release assay was developed to study owl monkey antibody-dependent cellular cytoxicity (ADCC) against RSV infected Hep-2 cells. Peripheral blood mononuclear cells from owl monkeys in the presence of RSV specific antibody caused lysis of RSV infected target cells. The effector cell for ADCC was found to be non-adherent to plastic. The owl monkey RSV ADCC system was found to closely parallel RSV specific ADCC observed with human effector cells. In addition, it was found that heterologous matches of human effector cells with monkey sera and monkey effector cells with human sera were equally efficient in mediating RSV specific ADCC. These studies demonstrate the value of the owl monkey as a model to study the pathogenesis or RSV infections.     

Dengue virus premembrane and membrane proteins elicit a protective immune response

We have constructed recombinant vaccinia viruses that express the premembrane (pre-M), membrane (M), or the cleaved, residual portion of pre-M (non-M) proteins of dengue 4 virus, or the pre-M, non-M, or envelope (E) proteins of dengue 2 virus, to evaluate their ability to induce protective immunity in mice. Cells infected with these recombinants make proteins of expected size. Mice immunized with recombinants expressing dengue 4 pre-M or M were protected against subsequent dengue 4 encephalitis challenge, but non-M was not protective. However, a recombinant that expressed both pre-M and E as a polyprotein gave solid protection, while the simultaneous administration of the two recombinants expressing pre-M and E gave a significant level of protection. Pre-M and M function as antigens eliciting a protective immune response, and the combination of pre-M plus E is more protective than E alone.     

Local and peripheral cell-mediated immune response to influenza virus in mice

Presentation of different influenza virus antigens generates different immune responses. Intranasal immunization with either live (VA) or formalin-inactivated (VF) A/PR/8/34 (HON1) influenza virus induced local as well as peripheral cell-mediated immune response (CMI), as evidenced by elevation in 3H-thymidine incorporation. Cell-mediated immune response was detected as soon as 24-48 hr following the application of VA and 4-5 days following VF. Cell-mediated immune response in both instances peaked on the 12th day and disappeared between 16 and 20 days after application. Local CMI response was threefold higher after immunization with VA (SI = 28.6) than with VF (SI = 9.4), while VF induced higher peripheral response (32.0 vs 17.7). The mononuclear cell population in the lungs increased, correlating with a rise in the stimulation index (SI). The percentage of IgA surface-bearing B lymphocytes was significantly higher following IN administration of VA, but not following VF instillation. This corroborated the finding that VF failed to induce local antibody response in the lungs in spite of its capacity to stimulate humoral antibody and CMI responses. Mice immunized intramuscularly with both viral preparations developed a fair humoral antibody response without detectable CMI (peripheral or local).     

Serum immune response to Shigella protein antigens in rhesus monkeys and humans infected with Shigella spp.

The serum antibody response to proteins encoded by the virulence-associated plasmid of Shigella flexneri was determined in monkeys challenged with virulent S. flexneri serotype 2a. With water-extractable antigen in an enzyme-linked immunosorbent assay, a significant increase in antibody titer against proteins from a plasmid-carrying, virulent strain of S. flexneri serotype 5 could be demonstrated in convalescent sera. There were minimal antibody titers against proteins from an avirulent (plasmid-free) organism. Previously identified plasmid-coded polypeptides a, b, c, and d were predominant antigens recognized by a majority of the convalescent sera in immunoblots. An additional 140-megadalton plasmid-coded polypeptide was also recognized by half of the sera. Convalescent serum from an infected monkey recognized antigens on the bacterial surface in several different plasmid-containing Shigella species and in an enteroinvasive Escherichia coli strain. A survey of sera obtained from children 5 to 10 years of age who had been infected with S. flexneri or S. sonnei revealed high enzyme-linked immunosorbent assay titers in both acute and convalescent sera against a water extract from a virulent Shigella strain. In contrast, children under 3 years of age had no antibody titer in either acute or convalescent sera against the virulence-associated shigella proteins, while 3- to 4-year-old children mounted an immune response against these proteins only in convalescence.     

Merozoite surface protein 1 of Plasmodium vivax induces a protective response against Plasmodium cynomolgi challenge in rhesus monkeys

The 42-kDa fragment of the merozoite surface protein 1 (MSP-1(42)) is a leading candidate for the development of a vaccine to control malaria. We previously reported a method for the production of Plasmodium vivax MSP-1(42) (PvMSP-1(42)) as a soluble protein (S. Dutta, L. W. Ware, A. Barbosa, C. F. Ockenhouse, and D. E. Lanar, Infect. Immun. 69:5464-5470, 2001). We report here a process to manufacture the same PvMSP-1(42) protein but as an insoluble inclusion body-derived protein which was then refolded in vitro. We compared the immunogenicity and protective efficacy of the soluble and refolded forms of PvMSP-1(42) protein by using a heterologous but closely related P. cynomolgi-rhesus monkey challenge model. As comparative controls we also expressed, purified, and immunized rhesus with the soluble and refolded forms of the P. cynomolgi MSP-1(42) (PcMSP-1(42)) proteins. All proteins induced equally high-titer, cross-reacting antibodies. Upon challenge with P. cynomolgi, none of the MSP-1(42)-vaccinated groups demonstrated sterile protection or a delay in the prepatent period. However, following an initial rise in parasitemia, all MSP-1-vaccinated animals had significantly lower parasite burdens as indicated by lower cumulative parasitemia, lower peak parasitemia, lower secondary peak parasitemia, and lower average daily parasitemia compared to the adjuvant control group (P < 0.05). Except the soluble PcMSP-1(42) group, monkeys in all other groups had fewer numbers of days with parasitemia of >10,000 parasites mm(-3). Interestingly, there was no significant difference in the level of partial protection observed in the homologous and heterologous groups in this challenge model. The soluble and refolded forms of PcMSP-1(42) and PvMSP-1(42) proteins also appeared to have a similar partially protective effect.     

Immunogenicity and protective efficacy in rhesus monkeys of a recombinant ORF2 protein from hepatitis E virus genotype 4

Several antigens derived from hepatitis E virus (HEV) genotype 1 strains have shown immunogenicity and efficacy against hepatitis E in primates and humans. However, the protective effect of a vaccine derived from HEV genotype 4 has not been studied. This study aimed to evaluate the immunogenicity and protective efficacy of the T1-ORF2 (56 kDa) capsid protein derived from HEV strain T1 (genotype 4) in rhesus monkeys. Two doses (40 μg) of alum-absorbed T1-ORF2 capsid protein were administered 4 weeks apart. Seroconversion occurred in all immunized monkeys 1–2 weeks after the first dose. The peak levels of anti-HEV IgG appeared at 2–3 weeks after the second dose and ranged from 5.7 to 196.0 U/ml. All monkeys showed an anamnestic antibody response to the second dose. Control monkeys immunized with saline remained negative for HEV antibodies throughout the pre-challenge period. The immunized monkeys were challenged intravenously with HEV genotypes 1 and 4. Monkeys immunized with T1-ORF2 were protected from infection and hepatitis after challenge with 5 × 10⁴ genome equivalents of HEV, regardless of the genotype. After challenge with 5 × 10⁵ genome equivalents of HEV genotype 4, the monkeys immunized with T1-ORF2 had a shorter period of raised alanine aminotransferase levels and a shorter duration of fecal shedding compared to control monkeys immunized with saline. In conclusion, these results suggest that, in rhesus monkeys, the T1-ORF2 capsid protein of HEV genotype 4 has similar cross-protective effects to other candidate vaccines derived from HEV genotype 1.     

Social separation and reunion affects immune system in juvenile rhesus monkeys.

Removal of juvenile rhesus monkeys from their natal social group to indoor individual caging resulted in increased basal cortisol secretion and significant decrements in the frequency of lymphoid subpopulations. Fourteen juvenile rhesus monkeys, which had never been removed from the group, were studied. Baseline immune and cortisol measurements were obtained before seven of the subjects were removed from social housing to standard individual cages. The remaining seven subjects, matched for age, sex, weight, and rank, remained in the social group throughout the study serving as controls. Blood samples were taken 24 hours after removal of the test subjects from the group and at specific intervals thereafter through 11 weeks. At 24 hours after the separation test subjects showed a significant increase in basal cortisol levels (40%) and a significant decrease in several immune parameters, with absolute numbers of total T cells declining 72 +/- 12%. Significant group differences in immune parameters persisted through 11 weeks. Eighteen weeks following removal, the test subjects were returned to the group which produced a cortisol rise in both test and controls at the 24-hour postreturn sample. Although there were no group differences in the frequency of lymphoid subsets 24 hours after return, some test subjects showed marked decrements which were inversely related to cortisol and were predicted by behavioral events. These data demonstrate that the removal of naive juvenile rhesus monkeys from their natal social group to individual indoor caging is a potent psychosocial stressor and that the behavioral interactions which characterize the return of the individual subjects to the natal group may predict physiological response.     

Liver disease in rhesus monkeys infected with simian immunodeficiency virus.

Rhesus monkeys infected with simian immunodeficiency virus (SIV) develop a syndrome very similar to patients with acquired immune deficiency (AIDS), including liver disease. This prospective study was undertaken to define the pathology, course, and pathogenesis of liver disease in 20 rhesus monkeys (Macaca mulatta) after intravenous inoculation with the standardized isolate SIV/DeltaB670. Tissue samples from liver and gallbladder between 2 and 24 weeks after inoculation were examined histologically and immunohistochemically for SIV gag protein p26, and by in situ hybridization with an SIV riboprobe. Histologically there was infiltration of portal tracts and around hepatic veins and venules by mononuclear inflammatory cells, focal bile duct damage, proliferation of bile ductules, and focal lobular inflammation as early as 2 weeks after infection. The severity and extent of these lesions were graded semiquantitatively and showed that bile duct damage and hepatic venulitis were the most significant changes. Simian immunodeficiency virus gag protein p26 and SIV RNA were detected in scattered mononuclear cells in portal tracts and sinusoids, but not in hepatocytes or bile duct epithelial cells. The data indicate that the liver is involved early during the course of SIV infection, followed by persistent changes until the terminal stage of the disease. Our findings suggest that the liver damage in SIV-infected rhesus monkeys is similar to the changes observed previously in AIDS patients.     

Oral immunization with Streptococcus mutants in rhesus monkeys and the development of immune response and dental caries.

The oral route of immunization with Streptococcus mutants was compared with the subcutaneous route in rhesus monkeys. Significant levels of serum IgG, IgM and IgA antibodies in Strep. mutans were elicited only in monkeys immunized subcutaneously. Similarly, the skin delayed hypersensitivity reaction to Strep. mutans was elicited only in the subcutaneously immunized monkeys. Oral immunization induced a modest increase in salivary IgA antibodies to Strep. mutans, though a slight increase in IgA antibodies was also found in the saliva of all other groups of immunized and control monkeys. A small though not significant reduction in dental caries was found in the monkeys immunized orally, whereas subcutaneous immunization with Strep. mutans consistently elicited a significant reduction in caries. Oral feeding of Strep. mutans failed to induce tolerance to a subsequent subcutaneous challenge by the same organism. Furthermore, sequential subcutaneous followed by oral immunization had little effect on the titre of salivary or serum antibodies.     

Indirect immunofluorescence, serum neutralization, and viremia responses of rhesus monkeys (Macaca mulatta) to Machupo virus

Although indirect immunofluorescent antibody tests (IFAT) have been developed for several arenaviruses, none has been applied to the rhesus monkey model for Bolivian hemorrhagic fever (caused by the arenavirus Machupo). We infected eight rhesus monkeys with Machupo virus and bled them weekly for determination of viremia and for serum antibody detection by IFAT and serum neutralization (SN) testing. Viremia peaked day 14 post-inoculation (PI), when two of eight animals had low IFAT titers. At day 21 PI, the six surviving monkeys had elevated IFAT titers and diminished viremias. SN titers were not observed until 28 days PI, when three of four survivors had low titers. Results of the IFAT were available more rapidly than the SN, and detected increased serum antibody titers earlier than the SN. Acetone fixation did not completely inactivate BHF antigen spot slides.