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Yellow fever virus (YFV) is a zoonotic pathogen re-emerging in parts of the world, causing a viral hemorrhagic fever associated with high mortality rates. While an effective vaccine is available, having an effective antiviral against YFV is critical against unexpected outbreaks, or when vaccination is not recommended. We have previously identified AT-281, the free base of AT-752, an orally available double prodrug of a guanosine nucleotide analog, as a potent inhibitor of YFV in vitro, with a 50% effective concentration (EC50) of 0.31 μM. In hamsters infected with YFV (Jimenez strain), viremia rose about 4 log10-fold and serum alanine aminotransferase (ALT) 2-fold compared to sham-infected animals. Treatment with 1000 mg/kg AT-752 for 7 days, initiated 4 h prior to viral challenge, reduced viremia to below the limit of detection by day 4 post infection (pi) and returned ALT to normal levels by day 6 pi. When treatment with AT-752 was initiated 2 days pi, the virus titer and ALT dropped >2 log10 and 53% by day 4 and 6 pi, respectively. In addition, at 21 days pi, 70–100% of the infected animals in the treatment groups survived compared to 0% of the untreated group (p, Author summary Yellow fever virus (YFV) is transmitted by mosquitoes, and its infection can lead to a lethal viral hemorrhagic fever associated with liver damage. While an effective vaccine is available, in places where the vaccination rate is low, in the event of an unexpected outbreak, or where vaccination is not recommended individually, having an effective antiviral treatment is critical. We previously reported that the nucleotide analog prodrug AT-752 potently inhibited the YFV in cultured cells. Here we showed that in hamsters infected with YFV, oral treatment with 1000 mg/kg AT-752 for 7 days reduced the production of infectious virus particles in the blood, and decreased serum alanine aminotransferase, a marker of liver damage, to levels measured in uninfected animals. In addition, at 21 days after infection, 70–100% of the infected animals in the treatment groups survived compared to 0% in the untreated group. Moreover, the amount of the active metabolite formed from AT-752 was highest in the livers and kidneys of the treated animals, organs that are targeted by the virus. These results suggest that AT-752 is a promising compound to develop for the treatment of YFV infection.

Background Hundreds of millions of people in poor countries continue to suffer from disease caused by bloodfeeding hookworms. While mice and rats are not reliably permissive hosts for any human hookworm species, adult Golden Syrian hamsters are fully permissive for the human and animal pathogen Ancylostoma ceylanicum. Similar to humans, hamsters may be infected with A. ceylanicum third-stage larvae orally or percutaneously. Oral infection typically leads to consistent worm yields in hamsters but may not accurately reflect the clinical and immunological manifestations of human infection resulting from skin penetration. Methodology/Principal findings In this study we compared host responses following percutaneous infection to those utilizing an established oral infection protocol. Infected hamsters exhibited a dose-dependent pathology, with 1000 percutaneous larvae (L3) causing anemia and adult worm recovery comparable to that of 50 orally administered L3. A delayed arrival and maturity of worms in the intestine was observed, as was variation in measured cellular immune responses. A long-term study found that the decline in blood hemoglobin was more gradual and did not reach levels as low, with the nadir of disease coming later in percutaneously infected hamsters. Both groups exhibited moderate growth delay, an effect that was more persistent in the percutaneously infected group. Fecal egg output also peaked later and at lower levels in the percutaneously infected animals. In contrast to orally infected hamsters, antibody titers to larval antigens continued to increase throughout the course of the experiment in the percutaneous group. Conclusions/Significance These results demonstrate that the route of infection with A. ceylanicum impacts disease pathogenesis, as well as humoral and cellular immune responses in an experimental setting. These data further validate the utility of the Golden Syrian hamster as a model of both oral and percutaneous infection with human hookworms., Author summary Hookworms are bloodfeeding intestinal parasites that represent an important cause of anemia and growth delay in children from Low and Middle Income Countries (LMICs). The study of hookworm is limited by the limited availability of animal models that accurately reproduce the clinical features of human infection. We report here a detailed description of hookworm infection in the hamster, with comparison of two routes of infection: oral vs percutaneous. The results demonstrate differences in the time course of infection and primary immune responses based on whether infection occurs orally or via skin penetration. These results build on the current understanding of hookworm pathogenesis and extend the utility of this important animal model of human disease.

Background Several infectious diseases are associated with hypothalamic-pituitary-adrenal (HPA) axis disorders by elevating circulating glucocorticoids (GCs), which are known to have an immunosuppressive potential. We conducted this study in golden hamsters, a suitable model for human visceral leishmaniasis (VL), to investigate the relationship of Leishmania (L.) infantum infection on cortisol production and VL severity. Methods L. infantum-infected (n = 42) and uninfected hamsters (n = 30) were followed-up at 30, 120, and 180 days post-infection (dpi). Plasma cortisol was analyzed by radioimmunoassay and cytokines, inducible nitric oxide synthase (iNOS), and arginase by RT-qPCR. Results All hamsters showed splenomegaly at 180 dpi. Increased parasite burden was associated with higher arginase expression and lower iNOS induction. Cortisol levels were elevated in infected animals in all-time points evaluated. Except for monocytes, all other leucocytes showed a strong negative correlation with cortisol, while transaminases were positively correlated. Immunological markers as interleukin (IL)-6, IL-1β, IL-10, and transforming growth-factor-β (TGF-β) were positively correlated to cortisol production, while interferon-γ (IFN-γ) presented a negative correlation. A network analysis showed cortisol as an important knot linking clinical status and immunological parameters. Conclusions These results suggest that L. infantum increases the systemic levels of cortisol, which showed to be associated with hematological, biochemical, and immunological parameters associated to VL severity., Author summary Visceral leishmaniasis (VL) is an infectious disease that is common in most tropical countries. VL has high morbidity and leads to death if not properly treated. In Brazil, Leishmania (Leishmania) infantum is the main causative agent of VL. Golden hamsters have proven to be a suitable model for VL. Despite the importance of hypothalamic-pituitary-adrenal (HPA) axis disturbances in infectious disease, few studies have addressed this issue in VL. In this study, we showed that L. infantum-infected hamsters present augmented levels of plasmatic cortisol in association with increased spleen parasite burden. Indeed, a strong positive correlation was observed between cortisol and biochemical parameters (AST/ALT/ALP) related to liver damage, as well as pro-inflammatory cytokines (IL-6 and IL-1β), anti-inflammatory cytokines (IL-10 and TGF-β), and the arginase enzyme that may favor the progression of infection. On the other side, cortisol was negatively correlated with leucocytes, except monocytes, and with IFN-γ and iNOS, which are involved in parasite-killing macrophage function. These results shed light on an unexplored aspect of VL pathogenesis, which is the importance of cortisol production in the disease-associated immune dysfunction.

The development of efficient vaccines against COVID-19 is an emergent need for global public health. The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a major target for the COVID-19 vaccine. To quickly respond to the outbreak of the SARS-CoV-2 pandemic, a nucleic acid-based vaccine is a novel option, beyond the traditional inactivated virus vaccine or recombinant protein vaccine. Here, we report a DNA vaccine containing the spike gene for delivery via electroporation. The spike genes of SARS-CoV and SARS-CoV-2 were codon optimized for mammalian cell expression and then cloned into mammalian cell expression vectors, called pSARS-S and pSARS2-S, respectively. Spike protein expression was confirmed by immunoblotting after transient expression in HEK293T cells. After immunization, sera were collected for antigen-specific antibody and neutralizing antibody titer analyses. We found that both pSARS-S and pSARS2-S immunization induced similar levels of antibodies against S2 of SARS-CoV-2. In contrast, only pSARS2-S immunization induced antibodies against the receptor-binding domain of SARS-CoV-2. We further found that pSARS2-S immunization, but not pSARS-S immunization, could induce very high titers of neutralizing antibodies against SARS-CoV-2. We further analyzed SARS-CoV-2 S protein-specific T cell responses and found that the immune responses were biased toward Th1. Importantly, pSARS2-S immunization in hamsters could induce protective immunity against SARS-CoV-2 challenge in vivo. These data suggest that DNA vaccination could be a promising approach for protecting against COVID-19., Author summary SARS-CoV-2 continues to threaten global health, and development of SARS-CoV-2 vaccine is an urgent priority to halt COVID-19 epidemic. Here, we developed DNA vaccines containing the spike gene from SARS-CoV-2 and delivered via electroporation. We observed that the DNA vaccine induced a long-lasting humoral response against SARS-CoV-2, and the elicited serum antibodies efficiently blocked the binding of SARS-CoV-2 RBD to its entry receptor (ACE2). Importantly, the DNA vaccine induced strong Th1-biased immune responses against SARS-CoV-2, which could provide protective effect with low side effects. Finally, we demonstrated that SARS-CoV-2 DNA vaccine conferred protective efficacy against SARS-CoV-2 infection in Syrian hamsters. Our findings suggested that DNA vaccination could be a useful approach for controlling COVID-19 pandemic in near future.

Background Opisthorchiasis is a hepatobiliary disease caused by flukes of the trematode family Opisthorchiidae. Opisthorchiasis can lead to severe hepatobiliary morbidity and is classified as a carcinogenic agent. Here we investigate the time-resolved metabolic response to Opisthorchis felineus infection in an animal model. Methodology Thirty golden hamsters were divided in three groups: severe infection (50 metacercariae/hamster), mild infection (15 metacercariae/hamster) and uninfected (vehicle-PBS) groups. Each group consisted of equal number of male and female animals. Plasma samples were collected one day before the infection and then every two weeks up to week 22 after infection. The samples were subjected to 1H Nuclear Magnetic Resonance (NMR) spectroscopy and multivariate statistical modelling. Principal findings The time-resolved study of the metabolic response to Opisthorchis infection in plasma in the main lines agrees with our previous report on urine data. The response reaches its peak around the 4th week of infection and stabilizes after the 10th week. Yet, unlike the urinary data there is no strong effect of the gender in the data and the intensity of infection is presented in the first two principal components of the PCA model. The main trends of the metabolic response to the infection in blood plasma are the transient depletion of essential amino acids and an increase in lipoprotein and cholesterol concentrations. Conclusions The time resolved metabolic signature of Opisthorchis infection in the hamster’s plasma shows a coherent shift in amino acids and lipid metabolism. Our work provides insight into the metabolic basis of the host response on the helminth infection., Author summary Opisthorchiasis is a parasitic infection caused by liver flukes of the Opisthorchiidae family. The liver fluke infection triggers development of hepatobiliary pathologies such as chronic forms of cholecystitis, cholangitis, pancreatitis, and cholelithiasis and increases the risk of intrahepatic cholangiocarcinoma. This manuscript is the second part of our outgoing project dedicated to a comprehensive description of the metabolic response to opisthorchiasis (more specifically Opisthorchis felineus) in an animal model. We show that the metabolic response in blood plasma is unfolding according to the same scenario as in urine, reaching its peak at the 4th week and stabilizing after the 10th week post-infection. Yet, unlike the response described in urine, the observed metabolic response in plasma is less gender specific. Moreover, the biochemical basis of the detected response in blood plasma is restricted to the remodeling of the lipid metabolism and the transient depletion of essential amino acids. Together with our first manuscript this report forms the first systematic description of the metabolic response on opisthorchiasis in an animal model using two easily accessible biofluids. Thus, this contribution provides novel results and fills an information gap still existing in the analytically driven characterization of the “Siberian liver fluke”, Opisthorchis felineus.

Background Leptospirosis has been described as a biphasic disease consisting of hematogenous dissemination to major organs in the acute phase and asymptomatic renal colonization in the chronic phase. Several observational studies have suggested an association between leptospirosis and chronic kidney disease (CKD). We investigated the dynamics of leptospires and histopathological changes in the kidney to understand the relationship between them, and also investigated the extent of renal dysfunction in the acute and chronic phases of leptospirosis using a hamster model. Findings Hamsters (n = 68) were subcutaneously infected with 1 × 104 cells of the Leptospira interrogans serovar Manilae strain UP-MMC-SM. A total of 53 infected hamsters developed fatal acute leptospirosis, and the remaining 15 hamsters recovered from the acute phase, 13 of which showed Leptospira colonization in the kidneys in the chronic phase. Five asymptomatic hamsters also had renal colonization in the chronic phase. Immunofluorescence staining showed that leptospires were locally distributed in the renal interstitium in the early acute phase and then spread continuously into the surrounding interstitium. The kidneys of the surviving hamsters in the chronic phase showed patchy lesions of atrophic tubules, a finding of chronic tubulointerstitial nephritis, which were substantially consistent with the distribution of leptospires in the renal interstitium. The degree of atrophic tubules in kidney sections correlated statistically with the serum creatinine level in the chronic phase (rs = 0.78, p = 0.01). Conclusion Subcutaneous infection with pathogenic leptospires could cause acute death or chronic leptospirosis in hamsters after surviving the acute phase. We suggest that the renal distribution of leptospires during the acute phase probably affected the extent of tubular atrophy, leading to CKD., Author summary Leptospirosis is the most widespread zoonotic disease, particularly in tropical and subtropical regions. Leptospires are transmitted to humans via contaminated urine, soil and water from carrier animals such as rodents. Human leptospirosis is mostly asymptomatic or a mild acute febrile illness, but some patients progress to Weil’s disease, characterized by kidney failure, jaundice, and pulmonary hemorrhage, and some of them die. To understand pathogen-host interactions in the kidney, a major target of leptospirosis, the authors collected kidneys and blood samples from the acute to chronic phase using a subcutaneously infected hamster model to compare the renal distribution of Leptospira with histopathological findings and renal function. The results demonstrated that the increase in antibody titers in the acute phase affected the outcome in the natural course of acute leptospirosis, and that the distribution of leptospires in the kidney during the acute phase may affect the decline of renal function during the chronic phase. It is possible that leptospirosis is one of the causes of unexplained renal dysfunction in tropical and subtropical regions.

Background Human hookworms (Necator americanus, Ancylostoma duodenale, and Ancylostoma ceylanicum) are intestinal blood-feeding parasites that infect ~500 million people worldwide and are among the leading causes of iron-deficiency anemia in the developing world. Drugs are useful against hookworm infections, but hookworms rapidly reinfect people, and the parasites can develop drug resistance. Therefore, having a hookworm vaccine would be of tremendous benefit. Methodology/Principal findings We investigated the vaccine efficacy in outbred Syrian hamsters of three A. ceylanicum hookworm antigen candidates from two classes of proteins previously identified as promising vaccine candidates. These include two intestinally-enriched, putatively secreted cathepsin B cysteine proteases (AceyCP1, AceyCPL) and one small Kunitz-type protease inhibitor (AceySKPI3). Recombinant proteins were produced in Pichia pastoris, and adsorbed to Alhydrogel. Recombinant AceyCPL (rAceyCPL)/Alhydrogel and rAceySKPI3/Alhydrogel induced high serum immunoglobulin G (IgG) titers in 8/8 vaccinates, but were not protective. rAceyCP1/Alhydrogel induced intermediate serum IgG titers in ~60% of vaccinates in two different trials. rAceyCP1 serum IgG responders had highly significantly decreased hookworm burdens, fecal egg counts and clinical pathology compared to Alhydrogel controls and nonresponders. Protection was highly correlated with rAceyCP1 serum IgG titer. Antisera from rAceyCP1 serum IgG responders, but not nonresponders or rAceyCPL/Alhydrogel vaccinates, significantly reduced adult A. ceylanicum motility in vitro. Furthermore, rAceyCP1 serum IgG responders had canonical Th2-specific recall responses (IL4, IL5, IL13) in splenocytes stimulated ex vivo. Conclusions/Significance These findings indicate that rAceyCP1 is a promising vaccine candidate and validates a genomic/transcriptomic approach to human hookworm vaccine discovery., Author summary Hookworms are voracious, blood-feeding, soil-transmitted nematode parasites. Adult hookworms infect the small intestine, causing iron-deficiency anemia and other complications. Hookworms are among the most disabling parasites of the developing world. Drugs are useful for controlling hookworm disease. However, because people often get reinfected rapidly and parasites can develop drug resistance, a vaccine that provides long-term protection would improve control and help lead to eradication. At present, there is no licensed hookworm vaccine, and progress towards a vaccine has been limited. We identified a cysteine protease in the intestine of the human hookworm Ancylostoma ceylanicum that is among the most strongly expressed genes during blood feeding and that may help digest blood and be essential for hookworm survival. Vaccination of hamsters with this cysteine protease gave high levels of protection when antigen-specific antibodies in the blood were induced. These antigen-specific antibodies also made hookworms less mobile in culture. This cysteine protease is a promising candidate for further investigation as a human hookworm vaccine antigen.

Background The liver fluke Opisthorchis viverrini infects several million people in Southeast Asia. Adult flukes live in the bile ducts of humans, where they cause hepatobiliary pathology, including cholangiocarcinoma. Here, we investigated the potential of extracellular vesicles (EVs) secreted by the fluke and defined recombinant proteins derived from EVs to generate protective immunity in a hamster vaccination-challenge model. Methodology/Principal findings EVs isolated from the excretory-secretory products of O. viverrini and two recombinant EV surface proteins encoding the large extracellular loops (LEL) of Ov-TSP-2 (rOv-TSP-2) and Ov-TSP-3 (rOv-TSP-3) were adjuvanted and used to vaccinate hamsters intraperitoneally followed by challenge infection with O. viverrini metacercariae. The number of adult flukes recovered from hamsters immunized with EVs, rOv-TSP-2, rOv-TSP-3 and rOv-TSP-2+rOv-TSP-3 were significantly reduced compared to control animals vaccinated with adjuvant alone. The number of eggs per gram feces was also significantly reduced in hamsters vaccinated with rOv-TSP-2 compared to controls, but no significant differences were found in the other groups. The average length of worms recovered from hamsters vaccinated with EVs, rOv-TSP-2 and rOv-TSP-3 was significantly shorter than that of worms recovered from the control group. Anti-EV IgG levels in serum and bile were significantly higher in hamsters vaccinated with EVs compared to control hamsters both pre- and post-challenge. In addition, levels of anti-rOv-TSP antibodies in the serum and bile were significantly higher than control hamsters both pre- and post-challenge. Finally, antibodies against rOv-TSP-2 and rOv-TSP-3 blocked uptake of EVs by human primary cholangiocyte in vitro, providing a plausible mechanism by which these vaccines exert partial efficacy and reduce the intensity of O. viverrini infection. Conclusion/Significance Liver fluke EVs and recombinant tetraspanins derived from the EV surface when administered to hamsters induce antibody responses that block EV uptake by target bile duct cells and exert partial efficacy and against O. viverrini challenge., Author summary Cholangiocarcinoma (CCA) is a significant public health problem in countries throughout Southeast Asia. In these areas CCA has a strong association with chronic infection with the food-borne liver fluke Opisthorchis viverrini. Current control of the infection relies on chemotherapy and health education, however these approaches are not sustainable in isolation. Hence, there is an urgent need for a vaccine against this neglected tropical disease. A vaccine against O. viverrini would confer anti-cancer protection in similar fashion to the acclaimed vaccine for human papillomavirus and cervical cancer. Toward this goal, secreted extracellular vesicles (EVs) of O. viverrini and recombinant proteins from the surface of EVs were generated and tested as vaccines in a hamster challenge model. Vaccination of hamsters with EVs and recombinant proteins induced production of antibodies in serum and bile, and those antibodies blocked uptake of EVs by primary bile duct cells in vitro. Challenge of vaccinated hamsters with infective stage flukes markedly reduced adult fluke recovery compared to the adjuvant control group. This is the first report of successful vaccination of hamsters with O. viverrini EVs and recombinant vesicle surface proteins, and provides proof-of-concept for development of subunit vaccines for this carcinogenic infection.

Rabies is a neglected zoonotic disease that is preventable in humans by appropriate post-exposure prophylaxis (PEP). However, current PEP relies on polyclonal immune globulin products purified from pooled human (HRIG) or equine (ERIG) plasma that are either in chronic shortage or in association with safety concerns. Here, we present the development of an antibody cocktail, SYN023, made of two novel monoclonal antibodies (MAb) CTB011 and CTB012 that could serve as safer and more cost-effective alternatives to the current RIG products. Both CTB011 and CTB012 are humanized MAbs that bind to non-overlapping epitopes on the rabies virus (RABV) glycoprotein (G) with sub-nanomolar affinities. Sequence analysis revealed that many of the critical residues in binding are highly conserved across different species of lyssaviruses. When combined at a 1:1 ratio, CTB011/CTB012 exhibited neutralization capabilities equivalent or superior to HRIG against 10 North American street RABV isolates in vitro and 15 prevalent Chinese RABV strains in animal models. Finally, SYN023, at a dosage of 0.03 mg/kg, was able to offer the same degree of protection as standard HRIG administration (20 IU/kg) in Syrian hamsters challenged with a highly virulent bat (Tadarida brasiliensis) RABV variant. Taken together, the high-potency and broad-spectrum neutralization demonstrated by SYN023 make it an effective candidate for human rabies PEP consideration., Author summary Rabies is a preventable viral disease and yet human rabies is still prevalent in many less-developed parts of the world. In those regions, the majority of human rabies deaths has been attributed to the low availability and poor accessibility of human antibodies (human immunoglobulin, HRIG), which can only be obtained from healthy individuals who volunteer to receive rabies vaccination. Although a cheaper alternative of equine origin (equine immunoglobulin, ERIG) is more readily available, it is not without safety risks and complications. HRIG and ERIG are effective in treating human rabies because they contain highly diverse antibodies that block different strains of rabies viruses from entering their pathogenic life cycle. To provide a similar spectrum of protection, we identified two antibodies, CTB011 and CTB012, which target conserved regions of the rabies viral particle across a wide range of strains. The 1:1 mixture of the two antibodies, SYN023, was demonstrated to have similar potency to HRIG in animal models. SYN023 therefore shows promise as a safer and more sustainable replacement of HRIG that can be massively produced to save lives.