Blocking NS3-NS4B interaction inhibits dengue virus in non-human primates.

Dengue is a major health threat and the number of symptomatic infections caused by the four dengue serotypes is estimated to be 96 million1 with annually around 10,000 deaths2. However, no antiviral drugs are available for the treatment or prophylaxis of dengue. We recently described the interaction between non-structural proteins NS3 and NS4B as a promising target for the development of pan-serotype dengue virus (DENV) inhibitors3. Here we present JNJ-1802-a highly potent DENV inhibitor that blocks the NS3-NS4B interaction within the viral replication complex. JNJ-1802 exerts picomolar to low nanomolar in vitro antiviral activity, a high barrier to resistance and potent in vivo efficacy in mice against infection with any of the four DENV serotypes. Finally, we demonstrate that the small-molecule inhibitor JNJ-1802 is highly effective against viral infection with DENV-1 or DENV-2 in non-human primates. JNJ-1802 has successfully completed a phase I first-in-human clinical study in healthy volunteers and was found to be safe and well tolerated4. These findings support the further clinical development of JNJ-1802, a first-in-class antiviral agent against dengue, which is now progressing in clinical studies for the prevention and treatment of dengue.

Longitudinal positron emission tomography and postmortem analysis reveals widespread neuroinflammation in SARS-CoV-2 infected rhesus macaques.

BACKGROUND: Coronavirus disease 2019 (COVID-19) patients initially develop respiratory symptoms, but they may also suffer from neurological symptoms. People with long-lasting effects after acute infections with severe respiratory syndrome coronavirus 2 (SARS-CoV-2), i.e., post-COVID syndrome or long COVID, may experience a variety of neurological manifestations. Although we do not fully understand how SARS-CoV-2 affects the brain, neuroinflammation likely plays a role. METHODS: To investigate neuroinflammatory processes longitudinally after SARS-CoV-2 infection, four experimentally SARS-CoV-2 infected rhesus macaques were monitored for 7 weeks with 18-kDa translocator protein (TSPO) positron emission tomography (PET) using [18F]DPA714, together with computed tomography (CT). The baseline scan was compared to weekly PET-CTs obtained post-infection (pi). Brain tissue was collected following euthanasia (50 days pi) to correlate the PET signal with TSPO expression, and glial and endothelial cell markers. Expression of these markers was compared to brain tissue from uninfected animals of comparable age, allowing the examination of the contribution of these cells to the neuroinflammatory response following SARS-CoV-2 infection. RESULTS: TSPO PET revealed an increased tracer uptake throughout the brain of all infected animals already from the first scan obtained post-infection (day 2), which increased to approximately twofold until day 30 pi. Postmortem immunohistochemical analysis of the hippocampus and pons showed TSPO expression in cells expressing ionized calcium-binding adaptor molecule 1 (IBA1), glial fibrillary acidic protein (GFAP), and collagen IV. In the hippocampus of SARS-CoV-2 infected animals the TSPO+ area and number of TSPO+ cells were significantly increased compared to control animals. This increase was not cell type specific, since both the number of IBA1+TSPO+ and GFAP+TSPO+ cells was increased, as well as the TSPO+ area within collagen IV+ blood vessels. CONCLUSIONS: This study manifests [18F]DPA714 as a powerful radiotracer to visualize SARS-CoV-2 induced neuroinflammation. The increased uptake of [18F]DPA714 over time implies an active neuroinflammatory response following SARS-CoV-2 infection. This inflammatory signal coincides with an increased number of TSPO expressing cells, including glial and endothelial cells, suggesting neuroinflammation and vascular dysregulation. These results demonstrate the long-term neuroinflammatory response following a mild SARS-CoV-2 infection, which potentially precedes long-lasting neurological symptoms.

Microbial molecule ingress promotes neuroinflammation and brain CCR5 expression in persons with HIV-associated neurocognitive disorders.

BACKGROUND: Systemic inflammation accompanies HIV-1 infection, resulting in microbial translocation from different tissues. We investigated interactions between lentivirus infections, neuroinflammation and microbial molecule presence in the brain. METHODS: Brain tissues from adult humans with (n = 22) and without HIV-1 (n = 11) infection as well as adult nonhuman primates (NHPs) with (n = 11) and without (n = 4) SIVmac251 infection were investigated by RT-PCR/ddPCR, immunofluorescence and western blotting. Studies of viral infectivity, host immune gene expression and viability were performed in primary human neural cells. FINDINGS: Among NHPs, SIV DNA quantitation in brain showed increased levels among animals with SIV encephalitis (n = 5) that was associated with bacterial genomic copy number as well as CCR5 and CASP1 expression in brain. Microbial DnaK and peptidoglycan were immunodetected in brains from uninfected and SIV-infected animals, chiefly in glial cells. Human microglia infected by HIV-1 showed increased p24 production after exposure to peptidoglycan that was associated CCR5 induction. HIV-1 Vpr application to human neurons followed by peptidoglycan exposure resulted in reduced mitochondrial function and diminished beta-III tubulin expression. In human brains, bacterial genome copies (250-550 copies/gm of tissue), were correlated with increased bacterial rRNA and GroEL transcript levels in patients with HIV-associated neurocognitive disorders (HAND). Glial cells displayed microbial GroEL and peptidoglycan immunoreactivity accompanied by CCR5 induction in brains from patients with HAND. INTERPRETATION: Increased microbial genomes and proteins were evident in brain tissues from lentivirus-infected humans and animals and associated with neurological disease. Microbial molecule translocation into the brain might exacerbate neuroinflammatory disease severity and represent a driver of lentivirus-associated brain disease.

Aerosolized pH1N1 influenza infection induces less systemic and local immune activation in the lung than combined intrabronchial, nasal and oral exposure in cynomolgus macaques.

Non-human primates form an important animal model for the evaluation of immunogenicity and efficacy of novel 'universal' vaccine candidates against influenza virus. However, in most studies a combination of intra-tracheal or intra-bronchial, oral and nasal virus inoculation is used with a standard virus dose of between 1 and 10 million tissue culture infective doses, which differs from typical modes of virus exposure in humans. This paper studies the systemic and local inflammatory and immune effects of aerosolized versus combined-route exposure to pandemic H1N1 influenza virus. In agreement with a previous study, both combined-route and aerosol exposure resulted in similar levels of virus replication in nose, throat and lung lavages. However, the acute release of pro-inflammatory cytokines and chemokines, acute monocyte activation in peripheral blood as well as increased cytokine production and T-cell proliferation in the lungs were only observed after combined-route infection and not after aerosol exposure. Longitudinal evaluation by computed tomography demonstrated persistence of lung lesions after resolution of the infection and a tendency for more lesions in the lower lung lobes after combined-route exposure versus upper and middle lung lobes after aerosol exposure. Computed tomography scores were observed to correlate with fever. In conclusion, influenza virus infection by aerosol exposure is accompanied by less immune-activation and inflammation in comparison with direct virus installation, despite similar levels of virus replication and development of lesions in the lungs.

Targeted Diet Modification Reduces Multiple Sclerosis-like Disease in Adult Marmoset Monkeys from an Outbred Colony.

Experimental autoimmune encephalomyelitis (EAE) in common marmosets is a translationally relevant model of the chronic neurologic disease multiple sclerosis. Following the introduction of a new dietary supplement in our purpose-bred marmoset colony, the percentage of marmosets in which clinically evident EAE could be induced by sensitization against recombinant human myelin oligodendrocyte glycoprotein in IFA decreased from 100 to 65%. The reduced EAE susceptibility after the dietary change coincided with reduced Callitrichine herpesvirus 3 expression in the colony, an EBV-related γ1-herpesvirus associated with EAE. We then investigated, in a controlled study in marmoset twins, which disease-relevant parameters were affected by the dietary change. The selected twins had been raised on the new diet for at least 12 mo prior to the study. In twin siblings reverted to the original diet 8 wk prior to EAE induction, 100% disease prevalence (eight out of eight) was restored, whereas in siblings remaining on the new diet the EAE prevalence was 75% (six out of eight). Spinal cord demyelination, a classical hallmark of the disease, was significantly lower in new-diet monkeys than in monkeys reverted to the original diet. In new-diet monkeys, the proinflammatory T cell response to recombinant human myelin oligodendrocyte glycoprotein was significantly reduced, and RNA-sequencing revealed reduced apoptosis and enhanced myelination in the brain. Systematic typing of the marmoset gut microbiota using 16S rRNA sequencing demonstrated a unique, Bifidobacteria-dominated composition, which changed after disease induction. In conclusion, targeted dietary intervention exerts positive effects on EAE-related parameters in multiple compartments of the marmoset's gut-immune-CNS axis.

Influenza virus infection as well as immunization with DNA encoding haemagglutinin protein induces potent antibody-dependent phagocytosis (ADP) and monocyte infection-enhancing responses in macaques.

Antibodies directed against the conserved regions within the influenza A haemagglutinin (HA) protein are detected at low frequency in humans. These antibodies display a broad reactivity against divergent influenza virus strains and could potentially offer broad protection. The in vivo protective effect of these antibodies is mainly mediated through Fc receptor effector functions. While antibody-dependent phagocytosis (ADP) of anti-HA antibodies has been demonstrated in human sera and sera from influenza virus-infected macaques, it is not known whether ADP can also be induced by vaccination and what the relative strength of ADP responses is in comparison to other antibody functions. Using a cohort of influenza virus-infected and immunized macaques, we demonstrate that infection as well as immunization with DNA-encoding HA induces high-titre ADP responses against HA, which are of potency 100-1000 times higher than virus inhibitory functions including antibody-dependent cell-mediated cytotoxicity (ADCC), virus neutralization (VN) and haemagglutinin inhibition (HAI). ADP activity was equally high against HA of heterologous influenza strains of the same subtype, in contrast to virus inhibitory functions, which were all greatly diminished. ADP titres against H5, representing a hetero-subtypic virus, were much lower. ADP was measured in THP-1 cells but was also observed in primary peripheral blood monocytes and neutrophils. Furthermore, at high serum dilution enhanced infection of both monocytes and myeloid dendritic cells (mDC) was observed. Hence, influenza virus infection as well as DNA-immunization against HA can induce high-titre ADP responses that can potentially enhance influenza virus infection of primary phagocytic and dendritic cells.

Role of microbial translocation in soluble CD14 up-regulation in HIV-, but not in HCV-, infected chimpanzees.

During human immunodeficiency virus (HIV) infection, soluble CD14 (sCD14) is up-regulated as a consequence of pathological disruption of the gut epithelial barrier, and subsequent increased microbial translocation. Also in hepatitis C virus (HCV)-infected patients with advanced liver fibrosis, increased levels of sCD14 have been reported. Since the liver plays an important role in clearance of translocated bacterial products, hepatic fibrosis may negatively affect clearance and thus contribute to higher sCD14 levels. Chimpanzees (Pan troglodytes) infected with HCV typically show no signs of liver fibrosis. Here, we have tested the hypothesis that increased levels of sCD14 occur in the absence of hepatic fibrosis or microbial translocation in chimpanzees chronically infected with HCV. sCD14 was up-regulated in both HIV/simian immunodeficiency virus (SIV)- and HCV-infected chimpanzees. In HIV/SIV-infected chimpanzees, intestinal fatty acid-binding protein, a marker for gut perturbation, lipopolysaccharide (LPS)-binding-protein and LPS core antibodies, confirm that sCD14 up-regulation was caused by increased microbial translocation. In HCV-infected chimpanzees, no evidence was found for increased microbial translocation despite up-regulation of sCD14. Additionally, the impact of liver fibrosis on microbial translocation was addressed by direct comparison of chimpanzees with a high HCV load and human patients with advanced fibrosis. These data suggest that only in a small minority of HCV patients, hepatic fibrosis corroborates microbial translocation.

A low dose of RBD and TLR7/8 agonist displayed on influenza virosome particles protects rhesus macaque against SARS-CoV-2 challenge.

Influenza virosomes serve as antigen delivery vehicles and pre-existing immunity toward influenza improves the immune responses toward antigens. Here, vaccine efficacy was evaluated in non-human primates with a COVID-19 virosome-based vaccine containing a low dose of RBD protein (15 µg) and the adjuvant 3M-052 (1 µg), displayed together on virosomes. Vaccinated animals (n = 6) received two intramuscular administrations at week 0 and 4 and challenged with SARS-CoV-2 at week 8, together with unvaccinated control animals (n = 4). The vaccine was safe and well tolerated and serum RBD IgG antibodies were induced in all animals and in the nasal washes and bronchoalveolar lavages in the three youngest animals. All control animals became strongly sgRNA positive in BAL, while all vaccinated animals were protected, although the oldest vaccinated animal (V1) was transiently weakly positive. The three youngest animals had also no detectable sgRNA in nasal wash and throat. Cross-strain serum neutralizing antibodies toward Wuhan-like, Alpha, Beta, and Delta viruses were observed in animals with the highest serum titers. Pro-inflammatory cytokines IL-8, CXCL-10 and IL-6 were increased in BALs of infected control animals but not in vaccinated animals. Virosomes-RBD/3M-052 prevented severe SARS-CoV-2, as shown by a lower total lung inflammatory pathology score than control animals.

Vaccine-induced neutralizing antibody responses to seasonal influenza virus H1N1 strains are not enhanced during subsequent pandemic H1N1 infection.

The first exposure to influenza is presumed to shape the B-cell antibody repertoire, leading to preferential enhancement of the initially formed responses during subsequent exposure to viral variants. Here, we investigated whether this principle remains applicable when there are large genetic and antigenic differences between primary and secondary influenza virus antigens. Because humans usually have a complex history of influenza virus exposure, we conducted this investigation in influenza-naive cynomolgus macaques. Two groups of six macaques were immunized four times with influenza virus-like particles (VLPs) displaying either one (monovalent) or five (pentavalent) different hemagglutinin (HA) antigens derived from seasonal H1N1 (H1N1) strains. Four weeks after the final immunization, animals were challenged with pandemic H1N1 (H1N1pdm09). Although immunization resulted in robust virus-neutralizing responses to all VLP-based vaccine strains, there were no cross-neutralization responses to H1N1pdm09, and all animals became infected. No reductions in viral load in the nose or throat were detected in either vaccine group. After infection, strong virus-neutralizing responses to H1N1pdm09 were induced. However, there were no increases in virus-neutralizing titers against four of the five H1N1 vaccine strains; and only a mild increase was observed in virus-neutralizing titer against the influenza A/Texas/36/91 vaccine strain. After H1N1pdm09 infection, both vaccine groups showed higher virus-neutralizing titers against two H1N1 strains of intermediate antigenic distance between the H1N1 vaccine strains and H1N1pdm09, compared with the naive control group. Furthermore, both vaccine groups had higher HA-stem antibodies early after infection than the control group. In conclusion, immunization with VLPs displaying HA from antigenically distinct H1N1 variants increased the breadth of the immune response during subsequent H1N1pdm09 challenge, although this phenomenon was limited to intermediate antigenic variants.

Novel polyomaviruses in South American bats and their relationship to other members of the family Polyomaviridae.

Bats are the natural reservoir of a variety of viruses, including a polyomavirus (PyV) from a North American brown bat. We investigated 163 spleen samples from 22 bat species from French Guiana for the presence of PyVs. In total, we detected 25 PyV-positive animals belonging to nine different bat species. Phylogenetic analysis was performed on the genomes of eight representative PyVs, and showed that the bat PyVs form three distinct lineages within the genus Orthopolyomavirus and are genetically different from the previously described North American bat virus. Interestingly, two lineages cluster with PyVs found in chimpanzees, orangutans and gorillas. In addition, one group of bat PyVs is genetically related to the human Merkel cell polyomavirus.

Safety and immunogenicity of four-segmented Rift Valley fever virus in the common marmoset.

Rift Valley fever virus (RVFV) is an emerging mosquito-borne bunyavirus that is highly pathogenic to wild and domesticated ruminants, camelids, and humans. While animals are exclusively infected via mosquito bites, humans can also be infected via contact with contaminated tissues or blood. No human vaccine is available and commercialized veterinary vaccines do not optimally combine efficacy with safety. We previously reported the development of two novel live-attenuated RVF vaccines, created by splitting the M genome segment and deleting the major virulence determinant NSs. The vaccine candidates, referred to as the veterinary vaccine vRVFV-4s and the human vaccine hRVFV-4s, were shown to induce protective immunity in multiple species after a single vaccination. Anticipating accidental exposure of humans to the veterinary vaccine and the application of hRVFV-4s to humans, the safety of each vaccine was evaluated in the most susceptible nonhuman primate model, the common marmoset (Callithrix jacchus). Marmosets were inoculated with high doses of each vaccine and were monitored for clinical signs as well as for vaccine virus dissemination, shedding, and spreading to the environment. To accurately assess the attenuation of both vaccine viruses, separate groups of marmosets were inoculated with the parent wild-type RVFV strains. Both wild-type strains induced high viremia and disseminated to primary target organs, associated with mild-to-severe morbidity. In contrast, both vaccines were well tolerated with no evidence of dissemination and shedding while inducing potent neutralizing antibody responses. The results of the studies support the unprecedented safety profile of both vaccines for animals and humans.

Novel application of [18F]DPA714 for visualizing the pulmonary inflammation process of SARS-CoV-2-infection in rhesus monkeys (Macaca mulatta).

RATIONALE: The aim of this study was to investigate the application of [18F]DPA714 to visualize the inflammation process in the lungs of SARS-CoV-2-infected rhesus monkeys, focusing on the presence of pulmonary lesions, activation of mediastinal lymph nodes and surrounded lung tissue. METHODS: Four experimentally SARS-CoV-2 infected rhesus monkeys were followed for seven weeks post infection (pi) with a weekly PET-CT using [18F]DPA714. Two PET images, 10 min each, of a single field-of-view covering the chest area, were obtained 10 and 30 min after injection. To determine the infection process swabs, blood and bronchoalveolar lavages (BALs) were obtained. RESULTS: All animals were positive for SARS-CoV-2 in both the swabs and BALs on multiple timepoints pi. The initial development of pulmonary lesions was already detected at the first scan, performed 2-days pi. PET revealed an increased tracer uptake in the pulmonary lesions and mediastinal lymph nodes of all animals from the first scan obtained after infection and onwards. However, also an increased uptake was detected in the lung tissue surrounding the lesions, which persisted until day 30 and then subsided by day 37-44 pi. In parallel, a similar pattern of increased expression of activation markers was observed on dendritic cells in blood. PRINCIPAL CONCLUSIONS: This study illustrates that [18F]DPA714 is a valuable radiotracer to visualize SARS-CoV-2-associated pulmonary inflammation, which coincided with activation of dendritic cells in blood. [18F]DPA714 thus has the potential to be of added value as diagnostic tracer for other viral respiratory infections.

Brain Inflammation and Intracellular α-Synuclein Aggregates in Macaques after SARS-CoV-2 Infection.

SARS-CoV-2 causes acute respiratory disease, but many patients also experience neurological complications. Neuropathological changes with pronounced neuroinflammation have been described in individuals after lethal COVID-19, as well as in the CSF of hospitalized patients with neurological complications. To assess whether neuropathological changes can occur after a SARS-CoV-2 infection, leading to mild-to-moderate disease, we investigated the brains of four rhesus and four cynomolgus macaques after pulmonary disease and without overt clinical symptoms. Postmortem analysis demonstrated the infiltration of T-cells and activated microglia in the parenchyma of all infected animals, even in the absence of viral antigen or RNA. Moreover, intracellular α-synuclein aggregates were found in the brains of both macaque species. The heterogeneity of these manifestations in the brains indicates the virus' neuropathological potential and should be considered a warning for long-term health risks, following SARS-CoV-2 infection.

Poxvirus MVA Expressing SARS-CoV-2 S Protein Induces Robust Immunity and Protects Rhesus Macaques From SARS-CoV-2.

Novel safe, immunogenic, and effective vaccines are needed to control the COVID-19 pandemic, caused by SARS-CoV-2. Here, we describe the safety, robust immunogenicity, and potent efficacy elicited in rhesus macaques by a modified vaccinia virus Ankara (MVA) vector expressing a full-length SARS-CoV-2 spike (S) protein (MVA-S). MVA-S vaccination was well tolerated and induced S and receptor-binding domain (RBD)-binding IgG antibodies and neutralizing antibodies against SARS-CoV-2 and several variants of concern. S-specific IFNγ, but not IL-4, -producing cells were also elicited. After SARS-CoV-2 challenge, vaccinated animals showed a significant strong reduction of virus loads in bronchoalveolar lavages (BAL) and decreased levels in throat and nasal mucosa. Remarkably, MVA-S also protected macaques from fever and infection-induced cytokine storm. Computed tomography and histological examination of the lungs showed reduced lung pathology in MVA-S-vaccinated animals. These findings favor the use of MVA-S as a potential vaccine for SARS-CoV-2 in clinical trials.

Molecular analysis of a novel simian virus 40 (SV40) type in rhesus macaques and evidence for double infections with the classical SV40 type.

The incidence of simian virus 40 (SV40) infections in rhesus macaques infected with simian-human immunodeficiency viruses (SHIV) and in uninfected animals was determined using PCR. Rates varied from 5% in peripheral blood mononuclear cells of uninfected monkeys to 19.6% in SHIV-infected macaques. Much higher detection rates, up to 75%, were found in lymph nodes and spleen samples of SHIV-infected animals. Sequence analysis of PCR amplicons revealed that they form two genetic clusters, one containing the majority of known SV40 strains and the other formed by variants with 7% genetic difference. Based on this difference, we propose two SV40 types: "type 1" or "classical type" for the majority of SV40 strains and "type 2" for the novel SV40 variants. The genome of one variant, SV40-Ri257, was completely sequenced and analyzed. The agnogene of SV40-Ri257 extends into the VP2 open reading frame and encodes a typical agnoprotein fused to a C-terminal hydrophobic region. The transcriptional control region (TCR) of SV40-Ri257 is the least conserved region compared to type 1 viruses. Particularly, the 3' end of the TCR, containing the early promoter and enhancer region, exhibits considerable variation. Further analysis of SHIV-infected macaques with type-specific PCRs revealed that the TCR of type 1 was completely conserved, whereas this region in type 2 varied considerably within the early enhancer region. We provide evidence here for the existence of a novel SV40 type in rhesus macaques and show that double infections with both types frequently occur.

Pinpointing the PRDM9-PRDM7 Gene Duplication Event During Primate Divergence.

Studies on the function of PRDM9 in model systems and its evolution during vertebrate divergence shed light on the basic molecular mechanisms of hybrid sterility and its evolutionary consequences. However, information regarding PRDM9-homolog, PRDM7, whose origin is placed in the primate evolutionary tree, as well as information about the fast-evolving DNA-binding zinc finger array of strepsirrhine PRDM9 are scarce. Thus, we aimed to narrow down the date of the duplication event leading to the emergence of PRDM7 during primate evolution by comparing the phylogenetic tree reconstructions of representative primate samples of PRDM orthologs and paralogs. To confirm our PRDM7 paralogization pattern, database-deposited sequences were used to test the presence/absence patterns expected from the paralogization timing. In addition, we extended the existing phylogenetic tree of haplorrhine PRDM9 zinc fingers with their strepsirrhine counterparts. The inclusion of strepsirrhine zinc fingers completes the PRDM9 primate phylogeny. Moreover, the updated phylogeny of PRDM9 zinc fingers showed distinct clusters of strepsirrhine, tarsier, and anthropoid degenerated zinc fingers. Here, we show that PRDM7 emerged on the branch leading to the most recent common ancestor of catarrhines; therefore, its origin is more recent than previously expected. A more detailed character evolutionary study suggests that PRDM7 may have evolved differently in Cercopithecoidea as compared to Hominoidea: it lacks the first four exons in Old World monkeys orthologs and exon 10 in Papionini orthologs. Dating the origin of PRDM7 is essential for further studies investigating why Hominoidea representatives need another putative histone methyltransferase in the testis.

TB and SIV Coinfection; a Model for Evaluating Vaccine Strategies against TB Reactivation in Asian Origin Cynomolgus Macaques: A Pilot Study Using BCG Vaccination.

This pilot study aimed to determine the utility of a cynomolgus macaque model of coinfection with simian immunodeficiency virus (SIV) for the assessment of vaccines designed to prevent reactivation of TB. Following infection caused by aerosol exposure to an ultralow dose of Mycobacterium tuberculosis (M. tb), data trends indicated that subsequent coinfection with SIVmac32H perturbed control of M. tb infection as evidenced by the increased occurrence of progressive disease in this group, higher levels of pathology and increased frequency of progressive tuberculous granulomas in the lung. BCG vaccination led to improved control of TB-induced disease and lower viral load in comparison to unvaccinated coinfected animals. The M. tb-specific IFNγ response after exposure to M. tb, previously shown to be associated with bacterial burden, was lower in the BCG-vaccinated group than in the unvaccinated groups. Levels of CD4+ and CD8+ T cells decreased in coinfected animals, with counts recovering more quickly in the BCG-vaccinated group. This pilot study provides proof of concept to support the use of the model for evaluation of interventions against reactivated/exacerbated TB caused by human immunodeficiency virus (HIV) infection.

Bronchoalveolar lavage affects thorax computed tomography of healthy and SARS-CoV-2 infected rhesus macaques (Macaca mulatta).

Medical imaging as method to assess the longitudinal process of a SARS-CoV-2 infection in non-human primates is commonly used in research settings. Bronchoalveolar lavage (BAL) is regularly used to determine the local virus production and immune effects of SARS-CoV-2 in the lower respiratory tract. However, the potential interference of those two diagnostic modalities is unknown in non-human primates. The current study investigated the effect and duration of BAL on computed tomography (CT) in both healthy and experimentally SARS-CoV-2-infected female rhesus macaques (Macaca mulatta). In addition, the effect of subsequent BALs was reviewed. Thorax CTs and BALs were obtained from four healthy animals and 11 experimentally SARS-CoV-2-infected animals. From all animals, CTs were obtained just before BAL, and 24 hours post-BAL. Additionally, from the healthy animals, CTs immediately after, and four hours post-BAL were obtained. Thorax CTs were evaluated for alterations in lung density, measured in Hounsfield units, and a visual semi-quantitative scoring system. An increase in the lung density was observed on the immediately post-BAL CT but resolved within 24 hours in the healthy animals. In the infected animals, a significant difference in both the lung density and CT score was still found 24 hours after BAL. Furthermore, the differences between time points in CT score were increased for the second BAL. These results indicate that the effect of BAL on infected lungs is not resolved within the first 24 hours. Therefore, it is important to acknowledge the interference between BAL and CT in rhesus macaques.

Aerosolized Exposure to H5N1 Influenza Virus Causes Less Severe Disease Than Infection via Combined Intrabronchial, Oral, and Nasal Inoculation in Cynomolgus Macaques.

Infection with highly pathogenic avian H5N1 influenza virus in humans often leads to severe respiratory disease with high mortality. Experimental infection in non-human primates can provide additional insight into disease pathogenesis. However, such a model should recapitulate the disease symptoms observed in humans, such as pneumonia and inflammatory cytokine response. While previous studies in macaques have demonstrated the occurrence of typical lesions in the lungs early after infection and a high level of immune activation, progression to severe disease and lethality were rarely observed. Here, we evaluated a routinely used combined route of infection via intra-bronchial, oral, and intra-nasal virus inoculation with aerosolized H5N1 exposure, with or without the regular collection of bronchoalveolar lavages early after infection. Both combined route and aerosol exposure resulted in similar levels of virus replication in nose and throat and similar levels of immune activation, cytokine, and chemokine release in the blood. However, while animals exposed to H5N1 by combined-route inoculation developed severe disease with high lethality, aerosolized exposure resulted in less lesions, as measured by consecutive computed tomography and less fever and lethal disease. In conclusion, not virus levels or immune activation, but route of infection determines fatal outcome for highly pathogenic avian H5N1 influenza infection.

A Bacterially-Expressed Recombinant Envelope Protein from Usutu Virus Induces Neutralizing Antibodies in Rabbits.

BACKGROUND: Recently, an emerging flavivirus, Usutu virus (USUV), has caused an epidemic among birds in Europe, resulting in a massive die-off in Eurasian blackbirds. Currently found only in Europe and Africa, it can be envisioned that Usutu virus will follow the path of other flaviviruses, like West Nile virus and Zika virus, and will spread via its mosquito vectors and bird hosts to other parts of the world. Several cases of human infections by Usutu virus have already been published. Anticipating this spread, development of an efficacious vaccine would be highly desirable. METHOD: This study describes the production in E. coli, purification, and refolding of a partial USUV envelope protein. Prior to immunization, the protein was characterized using size exclusion chromatography, transmission electron microscopy and dynamic light scattering, showing the limited presence of virus-like structures, indicating that the protein solution is probably a mixture of mono and multimeric envelope proteins. RESULTS: Immunizations of two rabbits with the refolded E-protein fraction, mixed with a strong adjuvant, resulted in the generation of neutralizing antibodies, as evidenced in an in vitro assay. DISCUSSION: The way forward towards a subunit vaccine against Usutu virus infection is discussed.