Animal models and SARS-CoV-2-induced pulmonary and neurological injuries.

Laboratory animals are essential mainly for experiments aiming to study pathogenesis and evaluate antivirals and vaccines against emerging human infectious diseases. Preclinical studies of coronavirus disease 19 (COVID-19) pathogenesis have used several animal species as models: transgenic human ACE2 mice (K18 mice), inbred BALB/c or C57BL/6N mice, ferrets, minks, domestic cats and dogs, hamsters, and macaques. However, the choice of an animal model relies on several limitations. Besides the host susceptibility, the researcher's experience with animal model management and the correct interpretation of clinical and laboratory records are crucial to succeed in preclinical translational research. Here, we summarise pathological and clinical findings correlated with virological data and immunological changes observed from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) experimental infections using different well-established SARS-CoV-2 animal model species. This essay aims to critically evaluate the current state of animal model translation to clinical data, as described in the human SARS-CoV-2 infection.

Detection of post-vaccination enhanced dengue virus infection in macaques: An improved model for early assessment of dengue vaccines.

The need for improved dengue vaccines remains since the only licensed vaccine, Dengvaxia, shows variable efficacy depending on the infecting dengue virus (DENV) type, and increases the risk of hospitalization for severe dengue in children not exposed to DENV before vaccination. Here, we developed a tetravalent dengue purified and inactivated vaccine (DPIV) candidate and characterized, in rhesus macaques, its immunogenicity and efficacy to control DENV infection by analyzing, after challenge, both viral replication and changes in biological markers associated with dengue in humans. Although DPIV elicited cross-type and long-lasting DENV-neutralizing antibody responses, it failed to control DENV infection. Increased levels of viremia/RNAemia (correlating with serum capacity at enhancing DENV infection in vitro), AST, IL-10, IL-18 and IFN-γ, and decreased levels of IL-12 were detected in some vaccinated compared to non-vaccinated monkeys, indicating the vaccination may have triggered antibody-dependent enhancement of DENV infection. The dengue macaque model has been considered imperfect due to the lack of DENV-associated clinical signs. However, here we show that post-vaccination enhanced DENV infection can be detected in this model when integrating several parameters, including characterization of DENV-enhancing antibodies, viremia/RNAemia, and biomarkers relevant to dengue in humans. This improved dengue macaque model may be crucial for early assessment of efficacy and safety of future dengue vaccines.

Safety and immunogenicity of 17DD attenuated yellow fever vaccine in howler monkeys (Alouatta spp.).

BACKGROUND: Alouatta spp. are highly susceptible to yellow fever (YF) infection and develop an often fatal disease. The threat posed by an outbreak started in 2016 leads us to investigate vaccination as a potential tool in preventing YF in non-human primates (NHP). METHODS: Susceptible howler monkeys were immunized with three different concentrations of the human Brazilian commercial YF17DD vaccine. Post-vaccination viremia/RNAemia, immunogenicity, and safety were characterized. RESULTS: The vaccine did not produce YF clinical manifestations in any of the NHPs. After immunization, all animals seroconverted demonstrating the ability of the YF vaccine to induce humoral response in Alouatta species. CONCLUSIONS: The present work has demonstrated the safe and immunogenic profile of the existing YF 17DD vaccine in howler monkeys. This knowledge may support further studies with other susceptible monkey species and provide a possible solution for controlling epizootics and preventing the devastation of endangered species.

Cynomolgus monkeys (Macaca fascicularis) experimentally infected with B19V and hepatitis A virus: no evidence of the co-infection as a cause of acute liver failure.

This study was conducted to analyse the course and the outcome of the liver disease in the co-infected animals in order to evaluate a possible synergic effect of human parvovirus B19 (B19V) and hepatitis A virus (HAV) co-infection. Nine adult cynomolgus monkeys were inoculated with serum obtained from a fatal case of B19V infection and/or a faecal suspension of acute HAV. The presence of specific antibodies to HAV and B19V, liver enzyme levels, viraemia, haematological changes, and necroinflammatory liver lesions were used for monitoring the infections. Seroconversion was confirmed in all infected groups. A similar pattern of B19V infection to human disease was observed, which was characterised by high and persistent viraemia in association with reticulocytopenia and mild to moderate anaemia during the period of investigation (59 days). Additionally, the intranuclear inclusion bodies were observed in pro-erythroblast cell from an infected cynomolgus and B19V Ag in hepatocytes. The erythroid hypoplasia and decrease in lymphocyte counts were more evident in the co-infected group. The present results demonstrated, for the first time, the susceptibility of cynomolgus to B19V infection, but it did not show a worsening of liver histopathology in the co-infected group.

Infection by Brazilian and Dutch swine hepatitis E virus strains induces haematological changes in Macaca fascicularis.

BACKGROUND: Hepatitis E virus (HEV) has been described as an emerging pathogen in Brazil and seems to be widely disseminated among swine herds. An autochthonous human case of acute hepatitis E was recently reported. To obtain a better understanding of the phenotypic profiles of both human and swine HEV strains, a experimental study was conducted using the animal model, Macaca fascicularis. METHODS: Six cynomolgus monkeys (Macaca fascicularis) were inoculated intravenously with swine HEV genotype 3 that was isolated from naturally and experimentally infected pigs in Brazil and the Netherlands. Two other monkeys were inoculated with HEV genotype 3 that was recovered from Brazilian and Argentinean patients with locally acquired acute and fulminant hepatitis E. The haematological, biochemical, and virological parameters of all animals were monitored for 67 days. RESULTS: Subclinical hepatitis was observed in all monkeys after inoculation with HEV genotype 3 that was recovered from the infected swine and human patients. HEV RNA was detected in the serum and/or faeces of 6 out of the 8 cynomolgus monkeys between 5 and 53 days after inoculation. The mild inflammation of liver tissues and elevations of discrete liver enzymes were observed. Seroconversions to anti-HEV IgM and/or IgG were detected in 7 animals. Reactivities to anti-HEV IgA were also detected in the salivary samples of 3 animals. Interestingly, all of the infected monkeys showed severe lymphopenia and a trend toward monocytosis, which coincided with elevations in alanine aminotransferase and antibody titres. CONCLUSIONS: The ability of HEV to cross the species barrier was confirmed for both the swine (Brazilian and Dutch) and human (Argentinean) strains, thus reinforcing the zoonotic risk of hepatitis E in South America. Cynomolgus monkeys that were infected with HEV genotype 3 developed subclinical hepatitis that was associated with haematological changes. Haematological approaches should be considered in future studies of HEV infection.

Safety and Immunogenicity of an In Vivo Muscle Electroporation Delivery System for DNA-hsp65 Tuberculosis Vaccine in Cynomolgus Monkeys.

A Bacille Calmette-Guérin (BCG) is still the only licensed vaccine for the prevention of tuberculosis, providing limited protection against Mycobacterium tuberculosis infection in adulthood. New advances in the delivery of DNA vaccines by electroporation have been made in the past decade. We evaluated the safety and immunogenicity of the DNA-hsp65 vaccine administered by intramuscular electroporation (EP) in cynomolgus macaques. Animals received three doses of DNA-hsp65 at 30-day intervals. We demonstrated that intramuscular electroporated DNA-hsp65 vaccine immunization of cynomolgus macaques was safe, and there were no vaccine-related effects on hematological, renal, or hepatic profiles, compared to the pre-vaccination parameters. No tuberculin skin test conversion nor lung X-ray alteration was identified. Further, low and transient peripheral cellular immune response and cytokine expression were observed, primarily after the third dose of the DNA-hsp65 vaccine. Electroporated DNA-hsp65 vaccination is safe but provides limited enhancement of peripheral cellular immune responses. Preclinical vaccine trials with DNA-hsp65 delivered via EP may include a combination of plasmid cytokine adjuvant and/or protein prime-boost regimen, to help the induction of a stronger cellular immune response.

Disposition of antimony in rhesus monkeys infected with Leishmania braziliensis and treated with meglumine antimoniate.

Antimony (Sb) disposition and toxicity was evaluated in Leishmania braziliensis-infected monkeys (Macaca mulatta) treated with a 21-d course of low (LOW) or standard (STD) meglumine antimoniate (MA) dosage regimens (5 or 20 mg Sb(V)/kg body weight/d im). Antimony levels in biological matrices were determined by inductively coupled plasma mass spectrometry (ICPMS), while on-line ion chromatography coupled to ICPMS was used to separate and quantify Sb species in plasma. Nadir Sb levels rose steadily from 19.6 ± 4 and 65.1 ± 17.4 ng/g, 24 h after the first injection, up to 27.4 ± 5.8 and 95.7 ± 6.6 ng/g, 24 h after the 21st dose in LOW and SDT groups, respectively. Subsequently, Sb plasma levels gradually declined with a terminal elimination phase half-life of 35.8 d. Antimony speciation in plasma on posttreatment days 1-9 indicated that as total Sb levels declined, proportion of Sb(V) remained nearly constant (11-20%), while proportion of Sb(III) rose from 5% (d 1) to 50% (d 9). Plasma [Sb]/erythrocyte [Sb] ratio was >1 until 12 h after dosing and reversed thereafter. Tissue Sb concentrations (posttreatment days 55 and 95) were as follows: >1000 ng/g in thyroid, nails, liver, gall bladder and spleen; >200 and <1000 ng/g in lymph nodes, kidneys, adrenals, bones, skeletal muscles, heart and skin; and <200 ng/g in various brain structures, thymus, stomach, colon, pancreas. and teeth. Results from this study are therefore consistent with view that Sb(V) is reduced to Sb(III), the active form, within cells from where it is slowly eliminated. Localization of Sb active forms in the thyroid gland and liver and the pathophysiological consequences of marked Sb accumulation in these tissues warrant further studies.

Evaluation of Molecular Test for the Discrimination of "Naked" DNA from Infectious Parvovirus B19 Particles in Serum and Bone Marrow Samples.

Low levels of parvovirus B19 (B19V) DNA can be detected in the circulation and in different tissue of immunocompetent individuals for months or years, which has been linked to inflammatory diseases such as cardiomyopathy, rheumatoid arthritis, hepatitis, and vasculitis. However, the detection of B19V DNA does not necessarily imply that infectious virions are present. This study aimed to evaluate the method based on the Benzonase® treatment for differentiation between the infectious virions from "naked" DNA in serum and bone marrow (BM) samples to be useful for the B19V routine diagnosis. In addition, we estimated the period of viremia and DNAemia in the sera and bone marrow of nonhuman primates experimentally infected with B19V. Serum samples from ten patients and from four cynomolgus monkeys experimentally infected with B19V followed up for 60 days were used. Most of the human serum samples became negative after pretreatment; however, only decreased viral DNA loads were observed in four patients, indicating that these samples still contained the infectious virus. Reduced B19V DNA levels were observed in animals since 7th dpi. At approximately 45th dpi, B19V DNA levels were below 105 IU/mL after Benzonase® pretreatment, which was not a consequence of active B19V replication. The test based on Benzonase® pretreatment enabled the discrimination of "naked DNA" from B19V DNA encapsidated in virions. Therefore, this test can be used to clarify the role of B19V as an etiological agent associated with atypical clinical manifestations.

Experimental hepatitis A virus (HAV) infection in cynomolgus monkeys (Macaca fascicularis): evidence of active extrahepatic site of HAV replication.

This work studied the replication sites of hepatitis A virus (HAV) in cynomolgus monkeys (Macaca fascicularis) after intravenous inoculation. The cynomolgus monkeys were inoculated with the Brazilian hepatitis A virus strain (HAF-203). Monkeys were euthanized on days 15, 30, 45 and 60 postinoculation (pi). Liver samples, submandibular salivary gland, mesenteric lymph node and tonsils were removed for virological and pathological evaluation. Immunofluorescence analyses on liver and salivary gland sections using confocal laser scanning microscopy revealed the presence of HAV antigen (HAV Ag). The presence of HAV genome was monitored by real-time PCR. The HAV RNA was detected at 7 days postinoculation (dpi), concomitantly in serum, saliva and faeces. The highest HAV viral load was observed in faeces at 15 dpi (10(5) copies/ml), followed by serum viral load of 10(4) copies/ml at 20 dpi and saliva viral load of 10(3 )copies/ml at 7 dpi. The animals showed first histological and biochemical signs of hepatitis at 15 dpi. The HAV antigen (Ag) was present from day 7 until day 60 pi in the liver and salivary glands. The HAV replicative intermediate was also detected in the liver (4.5 x 10(4) copies/mg), salivary glands (1.9 x 10(3) copies/mg), tonsils (4.2 x 10(1) copies/mg) and lymph nodes (3.4 x 10(1) copies/mg). Our data demonstrated that the salivary gland as an extrahepatic site of early HAV replication could create a potential risk of saliva transmitted infection. In addition, the cynomolgus monkey was confirmed as a suitable model to study the pathogenesis of HAV human infection.

Sofosbuvir shows a protective effect against vertical transmission of Zika virus and the associated congenital syndrome in rhesus monkeys.

The outbreaks of Zika virus (ZIKV) infection in Brazil, 2015-2016, were associated with severe congenital malformations. Our translational study aimed to test the efficacy of the antiviral agent sofosbuvir (SOF) against vertical transmission of ZIKV and the associated congenital syndrome (CZS), using a rhesus monkey model. Eight pregnant macaques were successfully infected during the organogenesis phase with a Brazilian ZIKV strain; five of them received SOF from two to fifteen days post-infection. Both groups of dams showed ZIKV-associated clinical signals, detectable ZIKV RNA in several specimens, specific anti-ZIKV IgM and IgG antibodies, and maternal neutralizing antibodies. However, malformations occurred only among non-treated dam offspring. Compared to non-treated animals, all SOF-treated dams had a shorter ZIKV viremia and four of five neonates had undetectable ZIKV RNA in blood and tissue samples. These results support further clinical evaluations aiming for the prevention of CZS.

Evidences of HEV genotype 3 persistence and reactivity in liver parenchyma from experimentally infected cynomolgus monkeys (Macaca fascicularis).

Hepatitis E virus genotype 3 (HEV-3) is an emerging zoonotic pathogen, responsible for sporadic cases of acute hepatitis E worldwide. Primate models have proven to be an essential tool for the study of HEV pathogenesis. Here we describe the outcomes of HEV infection in Macaca fascicularis (cynomolgus) inoculated experimentally with genotype 3. Eight adult cynomolgus macaques were inoculated intravenously with HEV-3 viral particles isolated from swine and human samples. Liver, spleen, duodenum, gallbladder and bile were sequential assessed up to the end-point of this study, 67 days post-inoculation (dpi). Our previously published findings showed that biochemical parameters return gradually to baseline levels at 55 dpi, whereas anti-HEV IgM and HEV RNA become undetectable in the serum and feces of all animals, indicating a non-viremic phase of recovery. Nevertheless, at a later stage during convalescence (67 dpi), the presence of HEV-3 RNA and antigen persist in central organs, even after peripheral viral clearance. Our results show that two cynomolgus inoculated with swine HEV-3 (animals I3 and O1) presented persistence of HEV RNA low titers in liver, gallbladder and bile. At this same stage of infection, HEV antigen (HEV Ag) could be detected in all infected animals, predominantly in non-reactive Kupffer cells (CD68+iNOS-) and sinusoidal lining cells. Simultaneously, CD4+, CD3+CD4+, and CD3+CD8+ immune cells were identified in hepatic sinusoids and small inflammatory clusters of lobular mononuclear cells, at the end-point of this study. Inability of HEV clearance in humans can result in chronic hepatitis, liver cirrhosis, with subsequent liver failure requiring transplantation. The results of our study support the persistence of HEV-3 during convalescence at 67 dpi, with active immune response in NHP. We alert to the inherent risk of viral transmission through liver transplantation, even in the absence of clinical and biochemical signs of acute infection. Thus, besides checking conventional serological markers of HEV infection, we strongly recommend HEV-3 RNA and antigen detection in liver explants as public health measure to prevent donor-recipient transmission and spread of hepatitis E.

Plant-Produced Subunit Vaccine Candidates against Yellow Fever Induce Virus Neutralizing Antibodies and Confer Protection against Viral Challenge in Animal Models.

Yellow fever (YF) is a viral disease transmitted by mosquitoes and endemic mostly in South America and Africa with 20-50% fatality. All current licensed YF vaccines, including YF-Vax® (Sanofi-Pasteur, Lyon, France) and 17DD-YFV (Bio-Manguinhos, Rio de Janeiro, Brazil), are based on live attenuated virus produced in hens' eggs and have been widely used. The YF vaccines are considered safe and highly effective. However, a recent increase in demand for YF vaccines and reports of rare cases of YF vaccine-associated fatal adverse events have provoked interest in developing a safer YF vaccine that can be easily scaled up to meet this increased global demand. To this point, we have engineered the YF virus envelope protein (YFE) and transiently expressed it in Nicotiana benthamiana as a stand-alone protein (YFE) or as fusion to the bacterial enzyme lichenase (YFE-LicKM). Immunogenicity and challenge studies in mice demonstrated that both YFE and YFE-LicKM elicited virus neutralizing (VN) antibodies and protected over 70% of mice from lethal challenge infection. Furthermore, these two YFE-based vaccine candidates induced VN antibody responses with high serum avidity in nonhuman primates and these VN antibody responses were further enhanced after challenge infection with the 17DD strain of YF virus. These results demonstrate partial protective efficacy in mice of YFE-based subunit vaccines expressed in N. benthamiana. However, their efficacy is inferior to that of the live attenuated 17DD vaccine, indicating that formulation development, such as incorporating a more suitable adjuvant, may be required for product development.

Characterization of recent and minimally passaged Brazilian dengue viruses inducing robust infection in rhesus macaques.

The macaque is widely accepted as a suitable model for preclinical characterization of dengue vaccine candidates. However, the only vaccine for which both preclinical and clinical efficacy results were reported so far showed efficacy levels that were substantially different between macaques and humans. We hypothesized that this model's predictive capacity may be improved using recent and minimally passaged dengue virus isolates, and by assessing vaccine efficacy by characterizing not only the post-dengue virus challenge viremia/RNAemia but also the associated-cytokine profile. Ten recent and minimally passaged Brazilian clinical isolates from the four dengue virus serotypes were tested for their infectivity in rhesus macaques. For the strains showing robust replication capacity, the associated-changes in soluble mediator levels, and the elicited dengue virus-neutralizing antibody responses, were also characterized. Three isolates from dengue virus serotypes 1, 2 and 4 induced viremia of high magnitude and longer duration relative to previously reported viremia kinetics in this model, and robust dengue virus-neutralizing antibody responses. Consistent with observations in humans, increased MCP-1, IFN-γ and VEGF-A levels, and transiently decreased IL-8 levels were detected after infection with the selected isolates. These results may contribute to establishing a dengue macaque model showing a higher predictability for vaccine efficacy in humans.

Studies on argyrophilic nucleolar organizer regions (AgNORs) in a spontaneous mammary gland ductal carcinoma of a captive rhesus monkey.

A spontaneous mammary gland ductal carcinoma was diagnosed in a 13-year-old female captive rhesus monkey (Macaca mulatta). The expression of argyrophilic nucleolar organizer regions (AgNORs) was studied to investigate the correlation between the histologic invasiveness and cell proliferation activity assay for predicting the biologic behavior of this tumor type. The results of this study show that the AgNOR size in tumor cells reflect the degree of malignancy when compared with the pattern of peripheral blood lymphocytes of the same individual. This is the first study showing a significant AgNOR feature of a malignant breast tumor in a rhesus monkey and it longs to provide additional diagnostic tool in tumor pathology.

Animal models and SARS-CoV-2-induced pulmonary and neurological injuries

Laboratory animals are essential mainly for experiments aiming to study pathogenesis and evaluate antivirals and vaccines against emerging human infectious diseases. Preclinical studies of coronavirus disease 19 (COVID-19) pathogenesis have used several animal species as models: transgenic human ACE2 mice (K18 mice), inbred BALB/c or C57BL/6N mice, ferrets, minks, domestic cats and dogs, hamsters, and macaques. However, the choice of an animal model relies on several limitations. Besides the host susceptibility, the researcher's experience with animal model management and the correct interpretation of clinical and laboratory records are crucial to succeed in preclinical translational research. Here, we summarise pathological and clinical findings correlated with virological data and immunological changes observed from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) experimental infections using different well-established SARS-CoV-2 animal model species. This essay aims to critically evaluate the current state of animal model translation to clinical data, as described in the human SARS-CoV-2 infection.

Cynomolgus monkeys are successfully and persistently infected with hepatitis E virus genotype 3 (HEV-3) after long-term immunosuppressive therapy.

Epidemiological studies found that hepatitis E virus genotype 3 (HEV-3) infection was associated with chronic hepatitis and cirrhosis in immunocompromised patients. Our study aimed to investigate the relationship between the host immunosuppressive status and the occurrence of HEV-related chronic hepatitis. Here we describe a successful experimental study, using cynomolgus monkeys previously treated with tacrolimus, a potent calcineurin inhibitor immunosuppressant, and infected with a Brazilian HEV-3 strain isolated from naturally infected pigs. HEV infected monkeys were followed up during 160 days post infection (dpi) by clinical signs; virological, biochemical and haematological parameters; and liver histopathology. The tacrolimus blood levels were monitored throughout the experiment. Immunosuppression was confirmed by clinical and laboratorial findings, such as: moderate weight loss, alopecia, and herpes virus opportunistic infection. In this study, chronic HEV infection was characterized by the mild increase of liver enzymes serum levels; persistent RNA viremia and viral faecal shedding; and liver histopathology. Three out of four immunosuppressed monkeys showed recurrent HEV RNA detection in liver samples, evident hepatocellular ballooning degeneration, mild to severe macro and microvesicular steatosis (zone 1), scattered hepatocellular apoptosis, and lobular focal inflammation. At 69 dpi, liver biopsies of all infected monkeys revealed evident ballooning degeneration (zone 3), discrete hepatocellular apoptosis, and at most mild portal and intra-acinar focal inflammation. At 160 dpi, the three chronically HEV infected monkeys showed microscopic features (piecemeal necrosis) corresponding to chronic hepatitis in absence of fibrosis and cirrhosis in liver parenchyma. Within 4-months follow up, the tacrolimus-immunosuppressed cynomolgus monkeys infected with a Brazilian swine HEV-3 strain exhibited more severe hepatic lesions progressing to chronic hepatitis without liver fibrosis, similarly as shown in tacrolimus-immunosuppressed solid organ transplant (SOT) recipients. The cause-effect relationship between HEV infection and tacrolimus treatment was confirmed in this experiment.

Experimental leptospirosis in marmoset monkeys (Callithrix jacchus): a new model for studies of severe pulmonary leptospirosis.

Experimental infection of marmoset monkeys (Callithrix jacchus) with Leptospira interrogans serovar Copenhageni showed microscopic patterns of tissue reactions comparable to those seen in the severe forms of human leptospirosis, including intra-alveolar hemorrhage. The most impressive microscopic changes were seen in the lung and kidney of animals killed at days 6 and 12 after inoculation. There were extensive and irregular areas of hemorrhage predominating around main bronchial branches or diffusely spread to the pulmonary parenchyma, as well as severe tubulointerstitial nephritis. Antibody response detected by the microscopic agglutination test was quantitatively similar to those seen in humans and paralleled severity of tissue lesions. The distribution of leptospires or antigenic debris in infected tissues was observed by immunofluorescence and confocal laser scanning microscopy. Large numbers of typical leptospires were seen in the lumen of proximal renal tubules. Positive reactions showing antigenic debris were closely associated with sites of tissue damage.

Trypanosoma cruzi in marsupial didelphids (Philander frenata and Didelhis marsupialis): differences in the humoral immune response in natural and experimental infections.

Philander frenata and Didelphis marsupialis harbor parasitism by Trypanosoma cruzi without developing any apparent disease and on the contrary to D. marsupialis, P. frenata maintains parasitism by T. cruzi II subpopulations. Here we compared the humoral immune response of the two didelphids naturally and experimentally infected with T. cruzi II group, employing SDS-PAGE/Western blot techniques and by an Indirect immunofluorescence assay. We also studied the histopathological pattern of naturally and experimentally infected P. frenata with T. cruzi. P. frenata sera recognized more antigens than D. marsupialis, and the recognition pattern did not show any change over the course of the follow up of both didelphid species. Polypeptides of 66 and 90kDa were the most prominent antigens recognized by both species in the soluble and enriched membrane fractions. P. frenata recognized intensely also a 45kDa antigen. Our findings indicate that: 1) there were no quantitative or qualitative differences in the patent or subpatent phases in the recognition pattern of P. frenata; 2) the significant differences in the recognition pattern of parasitic antigens by P. frenata and D. marsupialis sera suggest that they probably "learned" to live in harmony with T. cruzi by different strategies; 3) although P. frenata do not display apparent disease, tissular lesions tended to be more severe than has been described in D. marsupialis; and 4) Both didelphids probably acquired infection by T. cruzi after their evolutionary divergence.

Clinical and parasitological protection in a Leishmania infantum-macaque model vaccinated with adenovirus and the recombinant A2 antigen.

BACKGROUND: Visceral leishmaniasis (VL) is a severe vector-born disease of humans and dogs caused by Leishmania donovani complex parasites. Approximately 0.2 to 0.4 million new human VL cases occur annually worldwide. In the new world, these alarming numbers are primarily due to the impracticality of current control methods based on vector reduction and dog euthanasia. Thus, a prophylactic vaccine appears to be essential for VL control. The current efforts to develop an efficacious vaccine include the use of animal models that are as close to human VL. We have previously reported a L. infantum-macaque infection model that is reliable to determine which vaccine candidates are most worthy for further development. Among the few amastigote antigens tested so far, one of specific interest is the recombinant A2 (rA2) protein that protects against experimental L. infantum infections in mice and dogs. METHODOLOGY/PRINCIPAL FINDINGS: Primates were vaccinated using three rA2-based prime-boost immunization regimes: three doses of rA2 plus recombinant human interleukin-12 (rhIL-12) adsorbed in alum (rA2/rhIL-12/alum); two doses of non-replicative adenovirus recombinant vector encoding A2 (Ad5-A2) followed by two boosts with rA2/rhIL-12/alum (Ad5-A2+rA2/rhIL12/alum); and plasmid DNA encoding A2 gene (DNA-A2) boosted with two doses of Ad5-A2 (DNA-A2+Ad5-A2). Primates received a subsequent infectious challenge with L. infantum. Vaccines, apart from being safe, were immunogenic as animals responded with increased pre-challenge production of anti-A2-specific IgG antibodies, though with some variability in the response, depending on the vaccine formulation/protocol. The relative parasite load in the liver was significantly lower in immunized macaques as compared to controls. Protection correlated with hepatic granuloma resolution, and reduction of clinical symptoms, particularly when primates were vaccinated with the Ad5-A2+rA2/rhIL12/alum protocol. CONCLUSIONS/SIGNIFICANCE: The remarkable clinical protection induced by A2 in an animal model that is evolutionary close to humans qualifies this antigen as a suitable vaccine candidate against human VL.

Inducible nitric oxide synthase in heart tissue and nitric oxide in serum of Trypanosoma cruzi-infected rhesus monkeys: association with heart injury.

BACKGROUND: The factors contributing to chronic Chagas' heart disease remain unknown. High nitric oxide (NO) levels have been shown to be associated with cardiomyopathy severity in patients. Further, NO produced via inducible nitric oxide synthase (iNOS/NOS2) is proposed to play a role in Trypanosoma cruzi control. However, the participation of iNOS/NOS2 and NO in T. cruzi control and heart injury has been questioned. Here, using chronically infected rhesus monkeys and iNOS/NOS2-deficient (Nos2(-/-)) mice we explored the participation of iNOS/NOS2-derived NO in heart injury in T. cruzi infection. METHODOLOGY: Rhesus monkeys and C57BL/6 and Nos2(-/-) mice were infected with the Colombian T. cruzi strain. Parasite DNA was detected by polymerase chain reaction, T. cruzi antigens and iNOS/NOS2(+) cells were immunohistochemically detected in heart sections and NO levels in serum were determined by Griess reagent. Heart injury was assessed by electrocardiogram (ECG), echocardiogram (ECHO), creatine kinase heart isoenzyme (CK-MB) activity levels in serum and connexin 43 (Cx43) expression in the cardiac tissue. RESULTS: Chronically infected monkeys presented conduction abnormalities, cardiac inflammation and fibrosis, which resembled the spectrum of human chronic chagasic cardiomyopathy (CCC). Importantly, chronic myocarditis was associated with parasite persistence. Moreover, Cx43 loss and increased CK-MB activity levels were primarily correlated with iNOS/NOS2(+) cells infiltrating the cardiac tissue and NO levels in serum. Studies in Nos2(-/-) mice reinforced that the iNOS/NOS2-NO pathway plays a pivotal role in T. cruzi-elicited cardiomyocyte injury and in conduction abnormalities that were associated with Cx43 loss in the cardiac tissue. CONCLUSION: T. cruzi-infected rhesus monkeys reproduce features of CCC. Moreover, our data support that in T. cruzi infection persistent parasite-triggered iNOS/NOS2 in the cardiac tissue and NO overproduction might contribute to CCC severity, mainly disturbing of the molecular pathway involved in electrical synchrony. These findings open a new avenue for therapeutic tools in Chagas' heart disease.