Cell-type deconvolution of bulk RNA-Seq from kidney using opensource bioinformatic tools.

Traditional bulk RNA-Seq pipelines do not assess cell-type composition within heterogeneous tissues. Therefore, it is difficult to determine whether conflicting findings among samples or datasets are the result of biological differences or technical differences due to variation in sample collections. This report provides a user-friendly, open source method to assess cell-type composition in bulk RNA-Seq datasets for heterogeneous tissues using published single cell (sc)RNA-Seq data as a reference. As an example, we apply the method to analysis of kidney cortex bulk RNA-Seq data from female (N=8) and male (N=9) baboons to assess whether observed transcriptome sex differences are biological or technical, i.e., variation due to ultrasound guided biopsy collections. We found cell-type composition was not statistically different in female versus male transcriptomes based on expression of 274 kidney cell-type specific transcripts, indicating differences in gene expression are not due to sampling differences. This method of cell-type composition analysis is recommended for providing rigor in analysis of bulk RNA-Seq datasets from complex tissues. It is clear that with reduced costs, more analyses will be done using scRNA-Seq; however, the approach described here is relevant for data mining and meta analyses of the thousands of bulk RNA-Seq data archived in the NCBI GEO public database.

Inhibition of indoleamine dioxygenase leads to better control of tuberculosis adjunctive to chemotherapy.

The expression of indoleamine 2,3-dioxygenase (IDO), a robust immunosuppressant, is significantly induced in macaque tuberculosis (TB) granulomas, where it is expressed on IFN-responsive macrophages and myeloid-derived suppressor cells. IDO expression is also highly induced in human TB granulomas, and products of its activity are detected in patients with TB. In vivo blockade of IDO activity resulted in the reorganization of the granuloma with substantially greater T cells being recruited to the core of the lesions. This correlated with better immune control of TB and reduced lung M. tuberculosis burdens. To study if the IDO blockade strategy can be translated to a bona fide host-directed therapy in the clinical setting of TB, we studied the effect of IDO inhibitor 1-methyl-d-tryptophan adjunctive to suboptimal anti-TB chemotherapy. While two-thirds of controls and one-third of chemotherapy-treated animals progressed to active TB, inhibition of IDO adjunctive to the same therapy protected macaques from TB, as measured by clinical, radiological, and microbiological attributes. Although chemotherapy improved proliferative T cell responses, adjunctive inhibition of IDO further enhanced the recruitment of effector T cells to the lung. These results strongly suggest the possibility that IDO inhibition can be attempted adjunctive to anti-TB chemotherapy in clinical trials.

Blood pressure and the kidney cortex transcriptome response to high-sodium diet challenge in female nonhuman primates.

Blood pressure (BP) is influenced by genetic variation and sodium intake with sex-specific differences; however, studies to identify renal molecular mechanisms underlying the influence of sodium intake on BP in nonhuman primates (NHP) have focused on males. To address the gap in our understanding of molecular mechanisms regulating BP in female primates, we studied sodium-naïve female baboons (n = 7) fed a high-sodium (HS) diet for 6 wk. We hypothesized that in female baboons variation in renal transcriptional networks correlates with variation in BP response to a high-sodium diet. BP was continuously measured for 64-h periods throughout the study by implantable telemetry devices. Sodium intake, blood samples for clinical chemistries, and ultrasound-guided kidney biopsies were collected before and after the HS diet for RNA-Seq and bioinformatic analyses. We found that on the LS diet but not the HS diet, sodium intake and serum 17 ß-estradiol concentration correlated with BP. Furthermore, kidney transcriptomes differed by diet-unbiased weighted gene coexpression network analysis revealed modules of genes correlated with BP on the HS diet but not the LS diet. Our results showed variation in BP on the HS diet correlated with variation in novel kidney gene networks regulated by ESR1 and MYC; i.e., these regulators have not been associated with BP regulation in male humans or rodents. Validation of the mechanisms underlying regulation of BP-associated gene networks in female NHP will inform better therapies toward greater precision medicine for women.

Isoniazid and rifapentine treatment effectively reduces persistent M. tuberculosis infection in macaque lungs.

A once-weekly oral dose of isoniazid and rifapentine for 3 months (3HP) is recommended by the CDC for treatment of latent tuberculosis infection (LTBI). The aim of this study is to assess 3HP-mediated clearance of M. tuberculosis bacteria in macaques with asymptomatic LTBI. Twelve Indian-origin rhesus macaques were infected with a low dose (~10 CFU) of M. tuberculosis CDC1551 via aerosol. Six animals were treated with 3HP and 6 were left untreated. The animals were imaged via PET/CT at frequent intervals. Upon treatment completion, all animals except 1 were coinfected with SIV to assess reactivation of LTBI to active tuberculosis (ATB). Four of 6 treated macaques showed no evidence of persistent bacilli or extrapulmonary spread until the study end point. PET/CT demonstrated the presence of significantly more granulomas in untreated animals relative to the treated group. The untreated animals harbored persistent bacilli and demonstrated tuberculosis (TB) reactivation following SIV coinfection, while none of the treated animals reactivated to ATB. 3HP treatment effectively reduced persistent infection with M. tuberculosis and prevented reactivation of TB in latently infected macaques.

Modeling SARS-CoV-2: Comparative Pathology in Rhesus Macaque and Golden Syrian Hamster Models.

Coronavirus disease 2019 (COVID-19) in humans has a wide range of presentations, ranging from asymptomatic or mild symptoms to severe illness. Suitable animal models mimicking varying degrees of clinical disease manifestations could expedite development of therapeutics and vaccines for COVID-19. Here we demonstrate that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection resulted in subclinical disease in rhesus macaques with mild pneumonia and clinical disease in Syrian hamsters with severe pneumonia. SARS-CoV-2 infection was confirmed by formalin-fixed, paraffin-embedded (FFPE) polymerase chain reaction (PCR), immunohistochemistry, or in situ hybridization. Replicating virus in the lungs was identified using in situ hybridization or virus plaque forming assays. Viral encephalitis, reported in some COVID-19 patients, was identified in one macaque and was confirmed with immunohistochemistry. There was no evidence of encephalitis in hamsters. Severity and distribution of lung inflammation were substantially more in hamsters compared with macaques and exhibited vascular changes and virus-induced cytopathic changes as seen in COVID-19 patients. Neither the hamster nor macaque models demonstrated evidence for multisystemic inflammatory syndrome (MIS). Data presented here demonstrate that macaques may be appropriate for mechanistic studies of mild asymptomatic COVID-19 pneumonia and COVID-19-associated encephalitis, whereas Syrian hamsters may be more suited to study severe COVID-19 pneumonia.

Antiretroviral therapy timing impacts latent tuberculosis infection reactivation in a Mycobacterium tuberculosis/SIV coinfection model.

Studies using the nonhuman primate model of Mycobacterium tuberculosis/simian immunodeficiency virus coinfection have revealed protective CD4+ T cell-independent immune responses that suppress latent tuberculosis infection (LTBI) reactivation. In particular, chronic immune activation rather than the mere depletion of CD4+ T cells correlates with reactivation due to SIV coinfection. Here, we administered combinatorial antiretroviral therapy (cART) 2 weeks after SIV coinfection to study whether restoration of CD4+ T cell immunity occurred more broadly, and whether this prevented reactivation of LTBI compared to cART initiated 4 weeks after SIV. Earlier initiation of cART enhanced survival, led to better control of viral replication, and reduced immune activation in the periphery and lung vasculature, thereby reducing the rate of SIV-induced reactivation. We observed robust CD8+ T effector memory responses and significantly reduced macrophage turnover in the lung tissue. However, skewed CD4+ T effector memory responses persisted and new TB lesions formed after SIV coinfection. Thus, reactivation of LTBI is governed by very early events of SIV infection. Timing of cART is critical in mitigating chronic immune activation. The potential novelty of these findings mainly relates to the development of a robust animal model of human M. tuberculosis/HIV coinfection that allows the testing of underlying mechanisms.

BNT162b vaccines protect rhesus macaques from SARS-CoV-2.

A safe and effective vaccine against COVID-19 is urgently needed in quantities that are sufficient to immunize large populations. Here we report the preclinical development of two vaccine candidates (BNT162b1 and BNT162b2) that contain nucleoside-modified messenger RNA that encodes immunogens derived from the spike glycoprotein (S) of SARS-CoV-2, formulated in lipid nanoparticles. BNT162b1 encodes a soluble, secreted trimerized receptor-binding domain (known as the RBD-foldon). BNT162b2 encodes the full-length transmembrane S glycoprotein, locked in its prefusion conformation by the substitution of two residues with proline (S(K986P/V987P); hereafter, S(P2) (also known as P2 S)). The flexibly tethered RBDs of the RBD-foldon bind to human ACE2 with high avidity. Approximately 20% of the S(P2) trimers are in the two-RBD 'down', one-RBD 'up' state. In mice, one intramuscular dose of either candidate vaccine elicits a dose-dependent antibody response with high virus-entry inhibition titres and strong T-helper-1 CD4+ and IFNγ+CD8+ T cell responses. Prime-boost vaccination of rhesus macaques (Macaca mulatta) with the BNT162b candidates elicits SARS-CoV-2-neutralizing geometric mean titres that are 8.2-18.2× that of a panel of SARS-CoV-2-convalescent human sera. The vaccine candidates protect macaques against challenge with SARS-CoV-2; in particular, BNT162b2 protects the lower respiratory tract against the presence of viral RNA and shows no evidence of disease enhancement. Both candidates are being evaluated in phase I trials in Germany and the USA1-3, and BNT162b2 is being evaluated in an ongoing global phase II/III trial (NCT04380701 and NCT04368728).

Responses to acute infection with SARS-CoV-2 in the lungs of rhesus macaques, baboons and marmosets.

Non-human primate models will expedite therapeutics and vaccines for coronavirus disease 2019 (COVID-19) to clinical trials. Here, we compare acute severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in young and old rhesus macaques, baboons and old marmosets. Macaques had clinical signs of viral infection, mild to moderate pneumonitis and extra-pulmonary pathologies, and both age groups recovered in two weeks. Baboons had prolonged viral RNA shedding and substantially more lung inflammation compared with macaques. Inflammation in bronchoalveolar lavage was increased in old versus young baboons. Using techniques including computed tomography imaging, immunophenotyping, and alveolar/peripheral cytokine response and immunohistochemical analyses, we delineated cellular immune responses to SARS-CoV-2 infection in macaque and baboon lungs, including innate and adaptive immune cells and a prominent type-I interferon response. Macaques developed T-cell memory phenotypes/responses and bystander cytokine production. Old macaques had lower titres of SARS-CoV-2-specific IgG antibody levels compared with young macaques. Acute respiratory distress in macaques and baboons recapitulates the progression of COVID-19 in humans, making them suitable as models to test vaccines and therapies.

Lethality of SARS-CoV-2 infection in K18 human angiotensin-converting enzyme 2 transgenic mice.

Vaccine and antiviral development against SARS-CoV-2 infection or COVID-19 disease would benefit from validated small animal models. Here, we show that transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) by the human cytokeratin 18 promoter (K18 hACE2) represent a susceptible rodent model. K18 hACE2 transgenic mice succumbed to SARS-CoV-2 infection by day 6, with virus detected in lung airway epithelium and brain. K18 ACE2 transgenic mice produced a modest TH1/2/17 cytokine storm in the lung and spleen that peaked by day 2, and an extended chemokine storm that was detected in both lungs and brain. This chemokine storm was also detected in the brain at day 6. K18 hACE2 transgenic mice are, therefore, highly susceptible to SARS-CoV-2 infection and represent a suitable animal model for the study of viral pathogenesis, and for identification and characterization of vaccines (prophylactic) and antivirals (therapeutics) for SARS-CoV-2 infection and associated severe COVID-19 disease.