Single-Cell Profiling of Ebola Virus Disease In Vivo Reveals Viral and Host Dynamics.

Ebola virus (EBOV) causes epidemics with high mortality yet remains understudied due to the challenge of experimentation in high-containment and outbreak settings. Here, we used single-cell transcriptomics and CyTOF-based single-cell protein quantification to characterize peripheral immune cells during EBOV infection in rhesus monkeys. We obtained 100,000 transcriptomes and 15,000,000 protein profiles, finding that immature, proliferative monocyte-lineage cells with reduced antigen-presentation capacity replace conventional monocyte subsets, while lymphocytes upregulate apoptosis genes and decline in abundance. By quantifying intracellular viral RNA, we identify molecular determinants of tropism among circulating immune cells and examine temporal dynamics in viral and host gene expression. Within infected cells, EBOV downregulates STAT1 mRNA and interferon signaling, and it upregulates putative pro-viral genes (e.g., DYNLL1 and HSPA5), nominating pathways the virus manipulates for its replication. This study sheds light on EBOV tropism, replication dynamics, and elicited immune response and provides a framework for characterizing host-virus interactions under maximum containment.

The Adjuvant Combination of dmLT and Monophosphoryl Lipid A Activates the Canonical, Nonpyroptotic NLRP3 Inflammasome in Dendritic Cells and Significantly Interacts to Expand Antigen-Specific CD4 T Cells.

Adjuvants are often essential additions to vaccines that enhance the activation of innate immune cells, leading to more potent and protective T and B cell responses. Only a few vaccine adjuvants are currently used in approved vaccine formulations in the United States. Combinations of one or more adjuvants have the potential to increase the efficacy of existing and next-generation vaccines. In this study, we investigated how the nontoxic double mutant Escherichia coli heat-labile toxin R192G/L211A (dmLT), when combined with the TLR4 agonist monophosphoryl lipid A (MPL-A), impacted innate and adaptive immune responses to vaccination in mice. We found that the combination of dmLT and MPL-A induced an expansion of Ag-specific, multifaceted Th1/2/17 CD4 T cells higher than that explained by adding responses to either adjuvant alone. Furthermore, we observed more robust activation of primary mouse bone marrow-derived dendritic cells in the combination adjuvant-treated group via engagement of the canonical NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome complex. This was marked by a multiplicative increase in the secretion of active IL-1ß that was independent of classical gasdermin D-mediated pyroptosis. Moreover, the combination adjuvant increased the production of the secondary messengers cAMP and PGE2 in dendritic cells. These results demonstrate how certain adjuvant combinations could be used to potentiate better vaccine responses to combat a variety of pathogens.

Ebola Virus Disease Features Hemophagocytic Lymphohistiocytosis/Macrophage Activation Syndrome in the Rhesus Macaque Model.

BACKGROUND: Ebola virus (EBOV) disease (EVD) is one of the most severe and fatal viral hemorrhagic fevers and appears to mimic many clinical and laboratory manifestations of hemophagocytic lymphohistiocytosis syndrome (HLS), also known as macrophage activation syndrome. However, a clear association is yet to be firmly established for effective host-targeted, immunomodulatory therapeutic approaches to improve outcomes in patients with severe EVD. METHODS: Twenty-four rhesus monkeys were exposed intramuscularly to the EBOV Kikwit isolate and euthanized at prescheduled time points or when they reached the end-stage disease criteria. Three additional monkeys were mock-exposed and used as uninfected controls. RESULTS: EBOV-exposed monkeys presented with clinicopathologic features of HLS, including fever, multiple organomegaly, pancytopenia, hemophagocytosis, hyperfibrinogenemia with disseminated intravascular coagulation, hypertriglyceridemia, hypercytokinemia, increased concentrations of soluble CD163 and CD25 in serum, and the loss of activated natural killer cells. CONCLUSIONS: Our data suggest that EVD in the rhesus macaque model mimics pathophysiologic features of HLS/macrophage activation syndrome. Hence, regulating inflammation and immune function might provide an effective treatment for controlling the pathogenesis of acute EVD.

Transgenic expression of a T cell epitope in Strongyloides ratti reveals that helminth-specific CD4+ T cells constitute both Th2 and Treg populations.

Helminths are distinct from microbial pathogens in both size and complexity, and are the likely evolutionary driving force for type 2 immunity. CD4+ helper T cells can both coordinate worm clearance and prevent immunopathology, but issues of T cell antigen specificity in the context of helminth-induced Th2 and T regulatory cell (Treg) responses have not been addressed. Herein, we generated a novel transgenic line of the gastrointestinal nematode Strongyloides ratti expressing the immunodominant CD4+ T cell epitope 2W1S as a fusion protein with green fluorescent protein (GFP) and FLAG peptide in order to track and study helminth-specific CD4+ T cells. C57BL/6 mice infected with this stable transgenic line (termed Hulk) underwent a dose-dependent expansion of activated CD44hiCD11ahi 2W1S-specific CD4+ T cells, preferentially in the lung parenchyma. Transcriptional profiling of 2W1S-specific CD4+ T cells isolated from mice infected with either Hulk or the enteric bacterial pathogen Salmonella expressing 2W1S revealed that pathogen context exerted a dominant influence over CD4+ T cell phenotype. Interestingly, Hulk-elicited 2W1S-specific CD4+ T cells exhibited both Th2 and Treg phenotypes and expressed high levels of the EGFR ligand amphiregulin, which differed greatly from the phenotype of 2W1S-specific CD4+ T cells elicited by 2W1S-expressing Salmonella. While immunization with 2W1S peptide did not enhance clearance of Hulk infection, immunization did increase total amphiregulin production as well as the number of amphiregulin-expressing CD3+ cells in the lung following Hulk infection. Altogether, this new model system elucidates effector as well as immunosuppressive and wound reparative roles of helminth-specific CD4+ T cells. This report establishes a new resource for studying the nature and function of helminth-specific T cells.

Salmonella Persistence and Host Immunity Are Dictated by the Anatomical Microenvironment.

The intracellular bacterial pathogen Salmonella is able to evade the immune system and persist within the host. In some cases, these persistent infections are asymptomatic for long periods and represent a significant public health hazard because the hosts are potential chronic carriers, yet the mechanisms that control persistence are incompletely understood. Using a mouse model of chronic typhoid fever combined with major histocompatibility complex (MHC) class II tetramers to interrogate endogenous, Salmonella-specific CD4+ helper T cells, we show that certain host microenvironments may favorably contribute to a pathogen's ability to persist in vivo We demonstrate that the environment in the hepatobiliary system may contribute to the persistence of Salmonella enterica subsp. enterica serovar Typhimurium through liver-resident immunoregulatory CD4+ helper T cells, alternatively activated macrophages, and impaired bactericidal activity. This contrasts with lymphoid organs, such as the spleen and mesenteric lymph nodes, where these same cells appear to have a greater capacity for bacterial killing, which may contribute to control of bacteria in these organs. We also found that, following an extended period of infection of more than 2 years, the liver appeared to be the only site that harbored Salmonella bacteria. This work establishes a potential role for nonlymphoid organ immunity in regulating chronic bacterial infections and provides further evidence for the hepatobiliary system as the site of chronic Salmonella infection.

CD4+ T cell persistence and function after infection are maintained by low-level peptide:MHC class II presentation.

CD4(+) memory-phenotype T cells decline over time when generated in response to acute infections cleared by other components of the immune system. Therefore, it was of interest to assess the stability of CD4(+) T cells during a persistent Salmonella infection, which is typical of persistent phagocytic infections that are controlled by this lymphocyte subset. We found that CD4(+) T cells specific for Salmonella peptide:MHC class II (MHCII) ligands were numerically stable for >1 y after initial oral infection. This stability was associated with peptide:MHCII-driven proliferation by a small number of T cells in the secondary lymphoid organs that harbored bacteria. The persistent population consisted of multifunctional Th1 cells that induced PD-1 and became exhausted when transferred to hosts expressing the specific peptide:MHCII ligand in all parts of the body. Thus, persistent infection of phagocytes produced a CD4(+) T cell population that was stably maintained by low-level peptide:MHCII presentation.

Vaccination with a single CD4 T cell peptide epitope from a Salmonella type III-secreted effector protein provides protection against lethal infection.

Salmonella infections affect millions worldwide and remain a significant cause of morbidity and mortality. It is known from mouse studies that CD4 T cells are essential mediators of immunity against Salmonella infection, yet it is not clear whether targeting CD4 T cell responses directly with peptide vaccines against Salmonella can be effective in combating infection. Additionally, it is not known whether T cell responses elicited against Salmonella secreted effector proteins can provide protective immunity against infection. In this study, we investigated both of these possibilities using prime-boost immunization of susceptible mice with a single CD4 T cell peptide epitope from Salmonella secreted effector protein I (SseI), a component of the Salmonella type III secretion system. This immunization conferred significant protection against lethal oral infection, equivalent to that conferred by whole heat-killed Salmonella bacteria. Surprisingly, a well-characterized T cell epitope from the flagellar protein FliC afforded no protection compared to immunization with an irrelevant control peptide. The protective response appeared to be most associated with polyfunctional CD4 T cells raised against the SseI peptide, since no antibodies were produced against any of the peptides and very little CD8 T cell response was observed. Overall, this study demonstrates that eliciting CD4 T cell responses against components of the Salmonella type III secretion system can contribute to protection against infection and should be considered in the design of future Salmonella subunit vaccines.

Temporal expression of bacterial proteins instructs host CD4 T cell expansion and Th17 development.

Pathogens can substantially alter gene expression within an infected host depending on metabolic or virulence requirements in different tissues, however, the effect of these alterations on host immunity are unclear. Here we visualized multiple CD4 T cell responses to temporally expressed proteins in Salmonella-infected mice. Flagellin-specific CD4 T cells expanded and contracted early, differentiated into Th1 and Th17 lineages, and were enriched in mucosal tissues after oral infection. In contrast, CD4 T cells responding to Salmonella Type-III Secretion System (TTSS) effectors steadily accumulated until bacterial clearance was achieved, primarily differentiated into Th1 cells, and were predominantly detected in systemic tissues. Thus, pathogen regulation of antigen expression plays a major role in orchestrating the expansion, differentiation, and location of antigen-specific CD4 T cells in vivo.

Safety and immunogenicity of a delayed booster dose of the rVSVΔG-ZEBOV-GP vaccine for prevention of Ebola virus disease: a multicentre, open-label, phase 2 randomised controlled trial.

BACKGROUND: rVSVΔG-ZEBOV-GP is the first approved vaccine with clinical efficacy against Ebola virus disease. Although a seroprotective threshold has not been defined for those at occupational risk of exposure, the current vaccine strategy is to attain a sustained high level of antibody titres. The aim of this trial was to explore the effects of delayed boosting upon both the height and duration of antibody titres following primary immunisation. METHODS: In this open-label phase 2 randomised controlled trial, we compared antibody titres at month 36 in participants who had received a homologous booster dose at month 18 following primary immunisation with those who had received no booster. From Oct 25, 2016, to Jan 29, 2020, healthy adults aged 18 years or older deemed at occupational risk of exposure to Ebola virus due to laboratory work, clinical duties, or travel to an active endemic region were recruited from four hospital clinics in the USA and one hospital clinic in Canada and received primary vaccination with 2×107 plaque-forming unit per mL of VSVΔG-ZEBOV-GP. 18 months later, individuals who consented and were still eligible were randomly assigned 1:1 to receive either a homologous booster dose or no booster. Study visits for safety and serial blood collections for antibody titres were done on enrolled participants at months 0, 1, 3, 6, 12, 18, 19, 24, 30, and 36. Through July, 2021, a web-based application was used for randomisation, including assignments with schedules for each of the five sites using mixed permuted blocks. The trial was not masked to participants or site staff. The primary endpoint was a comparison of geometric mean titres (GMTs) of anti-Ebola virus glycoprotein IgG antibody at month 36 (ie, 18 months after randomisation) for all randomly assigned participants who completed the 36 months of follow-up (primary analysis cohort). Investigators were aware of antibody titres from baseline (enrolment) through month 18 but were masked to summary data by randomisation group after month 18. This study is registered with ClinicalTrials.gov (NCT02788227). FINDINGS: Of the 248 participants who enrolled and received their primary immunisation, 114 proceeded to the randomisation step at month 18. The two randomisation groups were balanced: 57 participants (24 [42%] of whom were female; median age was 42 years [IQR 35-50]) were randomly assigned to the booster group and 57 (24 [42%] of whom were female; median age was 42 years [IQR 36-51]) to the no-booster group. Of those randomly assigned, 92 participants (45 in the booster group and 47 in the no-booster group) completed 36 months of follow-up. At 18 months after primary immunisation, GMTs in the no-booster group increased from a baseline of 10 ELISA units (EU)/mL (95% CI 7-14) to 1451 EU/mL (1118-1882); GMTs in the booster group increased from 9 EU/mL (6-16) to 1769 EU/mL (1348-2321). At month 19, GMTs were 31 408 EU/mL (23 181-42 554) for the booster group and 1406 EU/mL (1078-1833) for the no-booster group; at month 36, GMTs were 10 146 EU/mL (7960-12 933) for the booster group and 1240 EU/mL (984-1563) for the no-booster group. Accordingly, the geometric mean ratio (GMR) of antibody titres had increased almost 21-fold more in the booster versus no-booster group at 1 month after booster administration (GMR 20·6; 95% CI 18·2-23·0; p<0·0001) and was still over 7-fold higher at month 36 (GMR 7·8; 95% CI 5·5-10·2; p<0·0001). Consistent with previous reports of this vaccine's side-effects, transient mono-articular or oligo-articular arthritis was diagnosed in 18 (9%) of 207 primary vaccination recipients; after randomisation, arthritis was diagnosed in one (2%) of 57 participants in the no-booster group. No new cases of arthritis developed after booster administration. Four serious adverse events occurred following randomisation: one (epistaxis) in the booster group and three (gastrointestinal haemorrhage, prostate cancer, and tachyarrhythmia) in the no-booster group. None of the serious adverse events was judged attributable to the booster vaccination assignment. INTERPRETATION: In addition to no new safety concerns and in marked contrast to earlier trials evaluating short-term boosting, delaying a rVSVΔG-ZEBOV-GP booster until month 18 resulted in an increase in GMT that remained several-fold above the no-booster group GMT for at least 18 months. These findings could have implications for defining the optimal timing of booster doses as pre-exposure prophylaxis in populations at ongoing risk for Ebola virus exposure. FUNDING: The Division of Intramural Research and the Division of Clinical Research of the National Institute of Allergy and Infectious Diseases at the US National Institutes of Health, Canadian Immunization Research Network through the Public Health Agency of Canada, Canadian Institutes of Health Research, and the US Defense Threat Reduction Agency.

Longitudinal analyses using 18F-Fluorodeoxyglucose positron emission tomography with computed tomography as a measure of COVID-19 severity in the aged, young, and humanized ACE2 SARS-CoV-2 hamster models.

This study compared disease progression of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in three different models of golden hamsters: aged (≈60 weeks old) wild-type (WT), young (6 weeks old) WT, and adult (14-22 weeks old) hamsters expressing the human-angiotensin-converting enzyme 2 (hACE2) receptor. After intranasal (IN) exposure to the SARS-CoV-2 Washington isolate (WA01/2020), 2-deoxy-2-[fluorine-18]fluoro-D-glucose positron emission tomography with computed tomography (18F-FDG PET/CT) was used to monitor disease progression in near real time and animals were euthanized at pre-determined time points to directly compare imaging findings with other disease parameters associated with coronavirus disease 2019 (COVID-19). Consistent with histopathology, 18F-FDG-PET/CT demonstrated that aged WT hamsters exposed to 105 plaque forming units (PFU) developed more severe and protracted pneumonia than young WT hamsters exposed to the same (or lower) dose or hACE2 hamsters exposed to a uniformly lethal dose of virus. Specifically, aged WT hamsters presented with a severe interstitial pneumonia through 8 d post-exposure (PE), while pulmonary regeneration was observed in young WT hamsters at that time. hACE2 hamsters exposed to 100 or 10 PFU virus presented with a minimal to mild hemorrhagic pneumonia but succumbed to SARS-CoV-2-related meningoencephalitis by 6 d PE, suggesting that this model might allow assessment of SARS-CoV-2 infection on the central nervous system (CNS). Our group is the first to use (18F-FDG) PET/CT to differentiate respiratory disease severity ranging from mild to severe in three COVID-19 hamster models. The non-invasive, serial measure of disease progression provided by PET/CT makes it a valuable tool for animal model characterization.

Persistent intraocular Ebola virus RNA is associated with severe uveitis in a convalescent rhesus monkey.

Despite increasing evidence that uveitis is common and consequential in survivors of Ebola virus disease (EVD), the host-pathogen determinants of the clinical phenotype are undefined, including the pathogenetic role of persistent viral antigen, ocular tissue-specific immune responses, and histopathologic characterization. Absent sampling of human intraocular fluids and tissues, these questions might be investigated in animal models of disease; however, challenges intrinsic to the nonhuman primate model and the animal biosafety level 4 setting have historically limited inquiry. In a rhesus monkey survivor of experimental Ebola virus (EBOV) infection, we observed and documented the clinical, virologic, immunologic, and histopathologic features of severe uveitis. Here we show the clinical natural history, resultant ocular pathology, intraocular antigen-specific antibody detection, and persistent intraocular EBOV RNA detected long after clinical resolution. The association of persistent EBOV RNA as a potential driver of severe immunopathology has pathophysiologic implications for understanding, preventing, and mitigating vision-threatening uveitis in EVD survivors.

Bacterial-Derived Outer Membrane Vesicles are Potent Adjuvants that Drive Humoral and Cellular Immune Responses.

Discovery and development of novel adjuvants that can improve existing or next generation vaccine platforms have received considerable interest in recent years. In particular, adjuvants that can elicit both humoral and cellular immune responses would be particularly advantageous because the majority of licensed vaccines are formulated with aluminum hydroxide (alum) which predominantly promotes antibodies. We previously demonstrated that bacterial-derived outer membrane vesicles (OMV) possess inherent adjuvanticity and drive antigen-specific antibody and cellular immune responses to OMV components. Here, we investigated the ability of OMVs to stimulate innate and adaptive immunity and to function as a stand-alone adjuvant. We show that OMVs are more potent than heat-inactivated and live-attenuated bacteria in driving dendritic cell activation in vitro and in vivo. Mice immunized with OMVs admixed with heterologous peptides generated peptide-specific CD4 and CD8 T cells responses. Notably, OMV adjuvant induced much greater antibody and B cell responses to co-delivered ovalbumin compared to the responses elicited by the adjuvants alum and CpG DNA. Additionally, pre-existing antibodies raised against the OMVs did not impair OMV adjuvanticity upon repeat immunization. These results indicate that vaccines adjuvanted with OMVs elicit robust cellular and humoral immune responses, supporting further development of OMV adjuvant for use in next-generation vaccines.

An Outer Membrane Vesicle-Adjuvanted Oral Vaccine Protects Against Lethal, Oral Salmonella Infection.

Non-typhoidal salmonellosis, caused by Salmonella enterica serovar Typhimurium is a common fecal-oral disease characterized by mild gastrointestinal distress resulting in diarrhea, chills, fever, abdominal cramps, head and body aches, nausea, and vomiting. Increasing incidences of antibiotic resistant invasive non-typhoidal Salmonella infections makes this a global threat requiring novel treatment strategies including next-generation vaccines. The goal of the current study was to formulate a novel vaccine platform against Salmonella infection that could be delivered orally. To accomplish this, we created a Salmonella-specific vaccine adjuvanted with Burkholderia pseudomallei outer membrane vesicles (OMVs). We show that adding OMVs to a heat-killed oral Salmonella vaccine (HKST + OMVs) protects against a lethal, oral challenge with Salmonella. Further, we show that opsonizing anti-Salmonella antibodies are induced in response to immunization and that CD4 T cells and B cells can be induced when OMVs are used as the oral adjuvant. This study represents a novel oral vaccine approach to combatting the increasing problem of invasive Salmonella infections.

Rhesus Macaque CODEX Multiplexed Immunohistochemistry Panel for Studying Immune Responses During Ebola Infection.

Non-human primate (NHP) animal models are an integral part of the drug research and development process. For some biothreat pathogens, animal model challenge studies may offer the only possibility to evaluate medical countermeasure efficacy. A thorough understanding of host immune responses in such NHP models is therefore vital. However, applying antibody-based immune characterization techniques to NHP models requires extensive reagent development for species compatibility. In the case of studies involving high consequence pathogens, further optimization for use of inactivated samples may be required. Here, we describe the first optimized CO-Detection by indEXing (CODEX) multiplexed tissue imaging antibody panel for deep profiling of spatially resolved single-cell immune responses in rhesus macaques. This 21-marker panel is composed of a set of 18 antibodies that stratify major immune cell types along with a set three Ebola virus (EBOV)-specific antibodies. We validated these two sets of markers using immunohistochemistry and CODEX in fully inactivated Formalin-Fixed Paraffin-Embedded (FFPE) tissues from mock and EBOV challenged macaques respectively and provide an efficient framework for orthogonal validation of multiple antibody clones using CODEX multiplexed tissue imaging. We also provide the antibody clones and oligonucleotide tag sequences as a valuable resource for other researchers to recreate this reagent set for future studies of tissue immune responses to EBOV infection and other diseases.

Control of Persistent Salmonella Infection Relies on Constant Thymic Output Despite Increased Peripheral Antigen-Specific T Cell Immunity.

Recent thymic emigrants are the youngest subset of peripheral T cells and their involvement in combating persistent bacterial infections has not been explored. Here, we hypothesized that CD4+ recent thymic emigrants are essential immune mediators during persistent Salmonella infection. To test this, we thymectomized adult mice either prior to, or during, persistent Salmonella infection. We found that thymic output is crucial in the formation of protective immune responses during the early formation of a Salmonella infection but is dispensable once persistent Salmonella infection is established. Further, we show that thymectomized mice demonstrate increased infection-associated mortality and bacterial burdens. Unexpectedly, numbers of Salmonella-specific CD4+ T cells were significantly increased in thymectomized mice compared to sham control mice. Lastly, we found that T cells from thymectomized mice may be impaired in producing the effector cytokine IL-17 at early time points of infection, compared to thymically intact mice. Together, these results imply a unique role for thymic output in the formation of immune responses against a persistent, enteric pathogen.

A Lassa Fever Live-Attenuated Vaccine Based on Codon Deoptimization of the Viral Glycoprotein Gene.

Lassa virus (LASV) is endemic in Western Africa and is estimated to infect hundreds of thousands of individuals annually. A considerable number of these infections result in Lassa fever (LF), which is associated with significant morbidity and a case-fatality rate as high as 69% among hospitalized confirmed patients. U.S. Food and Drug Administration-approved LF vaccines are not available. Current antiviral treatment is limited to off-label use of a nucleoside analogue, ribavirin, that is only partially effective and associated with significant side effects. We generated and characterized a recombinant LASV expressing a codon-deoptimized (CD) glycoprotein precursor gene (GPC), rLASV-GPC/CD. Comparison of growth kinetics and peak titers showed that rLASV-GPC/CD is slightly attenuated in cell culture compared to wild-type (WT) recombinant LASV (rLASV-WT). However, rLASV-GPC/CD is highly attenuated in strain 13 and Hartley guinea pigs, as reflected by the absence of detectable clinical signs in animals inoculated with rLASV-GPC/CD. Importantly, a single subcutaneous dose of rLASV-GPC/CD provides complete protection against an otherwise lethal exposure to LASV. Our results demonstrate the feasibility of implementing a CD approach for developing a safe and effective LASV live-attenuated vaccine candidate. Moreover, rLASV-GPC/CD might provide investigators with a tool to safely study LASV outside maximum (biosafety level 4) containment, which could accelerate the elucidation of basic aspects of the molecular and cell biology of LASV and the development of novel LASV medical countermeasures.IMPORTANCE Lassa virus (LASV) infects several hundred thousand people in Western Africa, resulting in many lethal Lassa fever (LF) cases. Licensed LF vaccines are not available, and anti-LF therapy is limited to off-label use of the nucleoside analog ribavirin with uncertain efficacy. We describe the generation of a novel live-attenuated LASV vaccine candidate. This vaccine candidate is based on mutating wild-type (WT) LASV in a key region of the viral genome, the glycoprotein precursor (GPC) gene. These mutations do not change the encoded GPC but interfere with its production in host cells. This mutated LASV (rLASV-GPC/CD) behaves like WT LASV (rLASV-WT) in cell culture, but in contrast to rLASV-WT, does not cause disease in inoculated guinea pigs. Guinea pigs immunized with rLASV-GPC/CD were protected against an otherwise lethal exposure to WT LASV. Our results support the testing of this candidate vaccine in nonhuman primate models ofLF.

A Lassa Virus Live-Attenuated Vaccine Candidate Based on Rearrangement of the Intergenic Region.

Lassa virus (LASV) poses a significant public health problem within the regions of Lassa fever endemicity in Western Africa. LASV infects several hundred thousand individuals yearly, and a considerable number of Lassa fever cases are associated with high morbidity and lethality. No approved LASV vaccine is available, and current therapy is limited to an off-label usage of ribavirin that is only partially effective and associated with significant side effects. The impact of Lassa fever on human health, together with the limited existing countermeasures, highlights the importance of developing effective vaccines against LASV. Here, we present the development and characterization of a recombinant LASV (rLASV) vaccine candidate [rLASV(IGR/S-S)], which is based on the presence of the noncoding intergenic region (IGR) of the small (S) genome segment (S-IGR) in both large (L) and S LASV segments. In cultured cells, rLASV(IGR/S-S) was modestly less fit than wild-type rLASV (rLASV-WT). rLASV(IGR/S-S) was highly attenuated in guinea pigs, and a single subcutaneous low dose of the virus completely protected against otherwise lethal infection with LASV-WT. Moreover, rLASV(IGR/S-S) was genetically stable during serial passages in cultured cells. These findings indicate that rLASV(IGR/S-S) can be developed into a LASV live-attenuated vaccine (LAV) that has the same antigenic composition as LASV-WT and a well-defined mechanism of attenuation that overcomes concerns about increased virulence that could be caused by genetic changes in the LAV during multiple rounds of multiplication.IMPORTANCE Lassa virus (LASV), the causative agent of Lassa fever, infects several hundred thousand people in Western Africa, resulting in many lethal Lassa fever cases. No U.S. Food and Drug Administration-licensed countermeasures are available to prevent or treat LASV infection. We describe the generation of a novel LASV live-attenuated vaccine candidate rLASV(IGR/S-S), which is based on the replacement of the large genomic segment noncoding intergenic region (IGR) with that of the small genome segment. rLASV(IGR/S-S) is less fit in cell culture than wild-type virus and does not cause clinical signs in inoculated guinea pigs. Importantly, rLASV(IGR/S-S) protects immunized guinea pigs against an otherwise lethal exposure to LASV.

Characteristic and quantifiable COVID-19-like abnormalities in CT- and PET/CT-imaged lungs of SARS-CoV-2-infected crab-eating macaques (Macaca fascicularis).

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causing an exponentially increasing number of coronavirus disease 19 (COVID-19) cases globally. Prioritization of medical countermeasures for evaluation in randomized clinical trials is critically hindered by the lack of COVID-19 animal models that enable accurate, quantifiable, and reproducible measurement of COVID-19 pulmonary disease free from observer bias. We first used serial computed tomography (CT) to demonstrate that bilateral intrabronchial instillation of SARS-CoV-2 into crab-eating macaques (Macaca fascicularis) results in mild-to-moderate lung abnormalities qualitatively characteristic of subclinical or mild-to-moderate COVID-19 (e.g., ground-glass opacities with or without reticulation, paving, or alveolar consolidation, peri-bronchial thickening, linear opacities) at typical locations (peripheral>central, posterior and dependent, bilateral, multi-lobar). We then used positron emission tomography (PET) analysis to demonstrate increased FDG uptake in the CT-defined lung abnormalities and regional lymph nodes. PET/CT imaging findings appeared in all macaques as early as 2 days post-exposure, variably progressed, and subsequently resolved by 6-12 days post-exposure. Finally, we applied operator-independent, semi-automatic quantification of the volume and radiodensity of CT abnormalities as a possible primary endpoint for immediate and objective efficacy testing of candidate medical countermeasures.

Establishment of Chronic Typhoid Infection in a Mouse Carriage Model Involves a Type 2 Immune Shift and T and B Cell Recruitment to the Gallbladder.

Typhoid fever, caused primarily by Salmonella enterica serovar Typhi (S Typhi), is a life-threatening systemic disease responsible for significant morbidity and mortality worldwide. Three to 5% of individuals infected with S Typhi become chronic carriers due to bacterial persistence in the gallbladder. We have demonstrated that Salmonella forms biofilms on gallstones to establish gallbladder carriage. However, an in-depth molecular understanding of chronic carriage in the gallbladder, from the perspective of both the pathogen and host, is poorly defined. To examine the dynamics of the gallbladder in response to Salmonella infection, we performed transcriptional profiling in the mouse gallbladder at early (7 days) and chronic (21 days) time points. Transcriptome sequencing (RNA-Seq) revealed a shift from a Th1 proinflammatory response at 7 days postinfection (dpi) toward an anti-inflammatory Th2 response by 21 dpi, characterized by increased levels of immunoglobulins and the Th2 master transcriptional regulator, GATA3. Additionally, bioinformatic analysis predicted the upstream regulation of characteristic Th2 markers, including interleukin-4 (IL-4) and Stat6. Immunohistochemistry and fluorescence-activated cell sorter (FACS) analysis confirmed a significant increase in lymphocytes, including T and B cells, at 21 dpi in mice with gallstones. Interestingly, the levels of Salmonella-specific CD4 T cells were 10-fold higher in the gallbladder of mice with gallstones at 21 dpi. We speculate that the biofilm state allows Salmonella to resist the initial onslaught of the Th1 inflammatory response, while yet undefined events influence a switch in the host immunity toward a more permissive type 2 response, enabling the establishment of chronic infection.IMPORTANCE The existence of chronic typhoid carriers has been in the public eye for over 100 years in part because of the publicity around Typhoid Mary. Additionally, it has been known for decades that the gallbladder is the main site of persistence and recently that gallstones play a key role. Despite this, very little is known about the physiological conditions that allow Salmonella enterica serovar Typhi to persist in the gallbladder. In this study, we analyze the transcriptional profile of the gallbladder in a mouse model of chronic carriage. We found a shift from an early proinflammatory immune response toward a later anti-inflammatory response, which could explain the stalemate that allows Salmonella persistence. Interestingly, we found a 10-fold increase in the number of Salmonella-specific T cells in mice with gallstones. This work moves us closer to understanding the mechanistic basis of chronic carriage, with a goal toward eradication of the disease.

Salmonella infection: Interplay between the bacteria and host immune system.

Salmonella infection causes morbidity and mortality throughout the world with the host immune response varying depending on whether the infection is acute and limited, or systemic and chronic. Additionally, Salmonella bacteria have evolved multiple mechanisms to avoid or subvert immunity to its own benefit and often the anatomical location of infection plays a role in both the immune response and bacterial fate. Here, we provide an overview of the interplay between the immune system and Salmonella, while discussing how different host and bacterial factors influence the outcome of infection.