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The most commonly used animal models for evaluating the efficacy of HSV-2 candidate vaccines are mice and guinea pigs. While numerous HSV-2 vaccine candidates have been tested in these animals and were effective in reducing disease and mortality, these results did not predict the effectiveness of the vaccines in human trials. Infection of rhesus macaques rarely results in lesions or HSV-2 specific antibody responses. In seeking an animal model that better recapitulates human disease and that might be more predictive of the efficacy of prophylactic vaccines than mice and guinea pigs, we evaluated Cebus apella (C. apella), a New World primate, in an HSV-2 genital infection model. Infectious HSV-2 was cultured from vaginal swabs from all 4 animals for 9–14 days after intravaginal inoculation of HSV-2 seronegative monkeys. Two of 4 monkeys had vesicular lesions in the vagina or vulva. No neurological symptoms were noted. Recurrent lesions and HSV-2 DNA shedding after acute disease resolved was infrequent. UV irradiation of the genital area did not induce recurrent genital lesions or virus shedding. All 4 monkeys developed HSV-2 neutralizing antibodies as well as virus-specific CD4 and CD8 T cell responses. Reinfection of animals 15 to 19 months after primary infection did not result in lesions; animals had reduced virus shedding and a shorter duration of shedding compared with that during primary infection, suggesting that primary infection induced protective immunity. Primary fibroblasts from C. apella monkeys supported the growth of HSV-2 in vitro; in contrast, HSV-2 did not replicate above the titer of the input inoculum in fibroblasts from rhesus macaques. These observations suggest that the C. apella monkey has potential to serve as a model for evaluating the efficacy of prophylactic vaccines, antivirals, or monoclonal antibodies to HSV-2. Author summary: Herpes simplex virus 2 (HSV-2) is a cause of genital herpes, neonatal herpes, and herpes encephalitis. The virus persists in the nervous system for life and periodically reactivates resulting in recurrent genital lesions and can be transmitted to susceptible individuals. Currently there is no cure for the disease and no licensed vaccine to prevent infection. Numerous HSV-2 vaccine candidates have been tested in animal models, mostly in mice and guinea pigs, and vaccine efficacy observed in these animals has been poorly predictive for effectiveness of HSV-2 vaccines in humans. Thus, there is a need for an animal model that better recapitulates human disease and is more predictive for the efficacy of prophylactic vaccines. We evaluated an HSV-2 genital infection model using Cebus apella, a New World primate. Infectious HSV-2 was cultured from vaginal swabs from all 4 animals for 9–14 days after intravaginal inoculation. The animals developed symptoms similar to most humans with genital vesicular lesions and no neurological signs. They also developed antibody and T cell responses to HSV-2. Prior infection generally reduced reinfection. These observations suggest that Cebus apella monkeys have the potential to serve as a good model for evaluating the efficacy of prophylactic HSV-2 vaccines. [ABSTRACT FROM AUTHOR]

The mRNA-1273 vaccine is effective against SARS-CoV-2 and was granted emergency use authorization by the FDA. Clinical studies, however, cannot provide the controlled response to infection and complex immunological insight that are only possible with preclinical studies. Hamsters are the only model that reliably exhibits severe SARS-CoV-2 disease similar to that in hospitalized patients, making them pertinent for vaccine evaluation. We demonstrate that prime or prime-boost administration of mRNA-1273 in hamsters elicited robust neutralizing antibodies, ameliorated weight loss, suppressed SARS-CoV-2 replication in the airways, and better protected against disease at the highest prime-boost dose. Unlike in mice and nonhuman primates, low-level virus replication in mRNA-1273-vaccinated hamsters coincided with an anamnestic response. Single-cell RNA sequencing of lung tissue permitted high-resolution analysis that is not possible in vaccinated humans. mRNA-1273 prevented inflammatory cell infiltration and the reduction of lymphocyte proportions, but enabled antiviral responses conducive to lung homeostasis. Surprisingly, infection triggered transcriptome programs in some types of immune cells from vaccinated hamsters that were shared, albeit attenuated, with mock- vaccinated hamsters. Our results support the use of mRNA-1273 in a 2-dose schedule and provide insight into the potential responses within the lungs of vaccinated humans who are exposed to SARS-CoV-2., Introduction When the World Health Organization declared severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) a global pandemic in March 2020, phase I clinical trials on the most promising vaccine candidates [...]

Omicron SARS-CoV-2 variants escape vaccine-induced neutralizing antibodies and cause nearly all current COVID-19 cases. Here, we compared the efficacy of three booster vaccines against Omicron BA.5 challenge in rhesus macaques: mRNA-1273, the Novavax ancestral spike protein vaccine (NVX-CoV2373), or Omicron BA.1 spike protein version (NVX-CoV2515). All three booster vaccines induced a strong BA.1 cross-reactive binding antibody and changed immunoglobulin G (Ig) dominance from IgG1 to IgG4 in the serum. All three booster vaccines also induced strong and comparable neutralizing antibody responses against multiple variants of concern, including BA.5 and BQ.1.1, along with long-lived plasma cells in the bone marrow. The ratio of BA.1 to WA-1 spike–specific antibody-secreting cells in the blood was higher in NVX-CoV2515 animals compared with NVX-CoV2373 animals, suggesting a better recall of BA.1-specific memory B cells by the BA.1 spike–specific vaccine compared with the ancestral spike–specific vaccine. Further, all three booster vaccines induced low levels of spike-specific CD4 but not CD8 T cell responses in the blood. After challenge with SARS-CoV-2 BA.5 variant, all three vaccines showed strong protection in the lungs and controlled virus replication in the nasopharynx. In addition, both Novavax vaccines blunted viral replication in nasopharynx at day 2. The protection against SARS-CoV-2 BA.5 infection in the upper respiratory airways correlated with binding, neutralizing, and ADNP activities of the serum antibody. These data have important implications for COVID-19 vaccine development, because vaccines that lower nasopharyngeal virus may help to reduce transmission. [ABSTRACT FROM AUTHOR]

Autosomal dominant hyper IgE syndrome (AD-HIES), or Job's syndrome, is a primary immune deficiency caused by dominant-negative mutations in STAT3. Recurrent Staphylococcus aureus skin abscesses are a defining feature of this syndrome. A widely held hypothesis that defects in peripheral Th17 differentiation confer this susceptibility has never been directly evaluated. To assess the cutaneous immune response in AD-HIES, we induced suction blisters in healthy volunteers (HVs) and patients with AD-HIES and then challenged the wound with lethally irradiated bacteria. We show that cutaneous production of IL-17A and IL-17F was normal in patients with AD-HIES. Overproduction of TNF- [alpha] differentiated the responses in ADHIES from HVs. This was associated with reduced IL-10 family signaling in blister- infiltrating cells and defective epithelial cell function. Mouse models of AD-HIES recapitulated these aberrant epithelial responses to S. aureus and involved defective epithelial-to-mesenchymal transition (EMT) rather than a failure of bacterial killing. Defective responses in mouse models of AD-HIES and primary keratinocyte cultures from patients with AD-HIES could be reversed by TNF-[alpha] blockade and by drugs with reported modulatory effects on EMT. Our results identify these as potential therapeutic approaches in patients with ADHIES suffering S. aureus infections., Introduction Autosomal dominant hyper IgE syndrome (AD-HIES), also known as Job's syndrome, is a primary immune deficiency caused by dominant-negative loss-of-function mutations in the STAT3 gene. Recurrent Staphylococcus aureus skin [...]

Astroviruses (AstVs) can cause of severe infection of the central nervous system (CNS) in immunocompromised individuals. Here, we identified a human AstV of the VA1 genotype, HAstV-NIH, as the cause of fatal encephalitis in an immunocompromised adult. We investigated the cells targeted by AstV, neurophysiological changes, and host responses by analyzing gene expression, protein expression, and cellular morphology in brain tissue from three cases of AstV neurologic disease (AstV-ND). We demonstrate that neurons are the principal cells targeted by AstV in the brain and that the cerebellum and brainstem have the highest burden of infection. Detection of VA1 AstV in interconnected brain structures such as thalamus, deep cerebellar nuclei, Purkinje cells, and pontine nuclei indicates that AstV may spread between connected neurons transsynaptically. We found transcriptional dysregulation of neural functions and disruption of both excitatory and inhibitory synaptic innervation of infected neurons. Importantly, transcriptional dysregulation of neural functions occurred in fatal cases, but not in a patient that survived AstV-ND. We show that the innate, but not adaptive immune response was transcriptionally driving host defense in the brain of immunocompromised patients with AstV-ND. Both transcriptome and molecular pathology studies showed that most of the cellular changes were associated with CNS-intrinsic cells involved in phagocytosis and injury repair (microglia, perivascular/parenchymal border macrophages, and astrocytes), but not CNS-extrinsic cells (T and B cells), suggesting an imbalance of innate and adaptive immune responses to AstV infection in the brain as a result of the underlying immunodeficiencies. These results show that VA1 AstV infection of the brain in immunocompromised humans is associated with imbalanced host defense responses, disruption of neuronal somatodendritic compartments and synapses and increased phagocytic cellular activity. Improved understanding of the response to viral infections of the human CNS may provide clues for how to manipulate these processes to improve outcomes. Author summary: Immunocompromised individuals are at increased risk of severe viral infection of the central nervous system (CNS) due to a failure to contain infection before it reaches the brain. Here, we identified the VA1-clade astrovirus infection as the cause of fatal encephalitis in an immunocompromised patient and show that it targeted neurons in the brain. Study of brain tissues from three cases of astrovirus encephalitis in immunocompromised patients showed that the disease was associated with impairment of many functions of neurons and disruption of both excitatory and inhibitory synapses that form neuronal connections. Further analyses showed that the early innate, but not later adaptive immune response was driving antiviral host defense response in the brain. Activation of the CNS-resident cells important for phagocytosis, homeostasis, and repair (microglia, macrophages, and astrocytes) was evident, with little activity of the adaptive peripheral immune cells (T cells and B cells). These results demonstrate that the pathogenesis and outcome of astrovirus infection in the brain of patients with underlying immunodeficiencies is defined by an imbalance of different arms of the antiviral host defense system and disruption of functions of infected neurons. These findings suggest pathways that can be targeted to improve outcome of viral CNS infections. [ABSTRACT FROM AUTHOR]

The pediatric live-attenuated bovine/human parainfluenza virus type 3 (B/HPIV3)-vectored vaccine expressing the prefusion-stabilized SARS-CoV-2 spike (S) protein (B/HPIV3/S-2P) was previously evaluated in vitro and in hamsters. To improve its immunogenicity, we generated B/HPIV3/S-6P, expressing S further stabilized with 6 proline mutations (S-6P). Intranasal immunization of hamsters with B/HPIV3/S-6P reproducibly elicited significantly higher serum anti-S IgA/IgG titers than B/HPIV3/S-2P; hamster sera efficiently neutralized variants of concern (VoCs), including Omicron variants. B/HPIV3/S-2P and B/HPIV3/S-6P immunization protected hamsters against weight loss and lung inflammation following SARS-CoV-2 challenge with the vaccine-matched strain WA1/2020 or VoCs B.1.1.7/Alpha or B.1.351/Beta and induced near-sterilizing immunity. Three weeks post-challenge, B/HPIV3/S-2P- and B/HPIV3/S-6P-immunized hamsters exhibited a robust anamnestic serum antibody response with increased neutralizing potency to VoCs, including Omicron sublineages. B/HPIV3/S-6P primed for stronger anamnestic antibody responses after challenge with WA1/2020 than B/HPIV3/S-2P. B/HPIV3/S-6P will be evaluated as an intranasal vaccine to protect infants against both HPIV3 and SARS-CoV-2. Author summary: SARS-CoV-2 infects and causes disease in all age groups. While injectable SARS-CoV-2 vaccines are effective against severe COVID-19, they do not fully prevent SARS-CoV-2 replication and transmission. This study describes the preclinical comparison in hamsters of B/HPIV3/S-2P and B/HPIV3/S-6P, live-attenuated pediatric vector vaccine candidates expressing the "2P" prefusion stabilized version of the SARS-CoV-2 spike protein, or the further-stabilized "6P" version. B/HPIV3/S-6P induced significantly stronger anti-S serum IgA and IgG responses than B/HPIV3/S-2P. A single intranasal immunization with B/HPIV3/S-6P elicited broad systemic antibody responses in hamsters that efficiently neutralized the vaccine-matched isolate as well as variants of concern, including Omicron. B/HPIV3/S-6P immunization induced near-complete airway protection against the vaccine-matched SARS-CoV-2 isolate as well as two variants. Furthermore, following SARS-CoV-2 challenge, immunized hamsters exhibited strong anamnestic serum antibody responses. Based on these data, B/HPIV3/S-6P will be further evaluated in a phase I study. [ABSTRACT FROM AUTHOR]

Allergic diseases are a global health challenge. Individuals harboring loss-of-function variants in transforming growth factor–β receptor (TGFβR) genes have an increased prevalence of allergic disorders, including eosinophilic esophagitis. Allergic diseases typically localize to mucosal barriers, implicating epithelial dysfunction as a cardinal feature of allergic disease. Here, we describe an essential role for TGFβ in the control of tissue-specific immune homeostasis that provides mechanistic insight into these clinical associations. Mice expressing a TGFβR1 loss-of-function variant identified in atopic patients spontaneously develop disease that clinically, immunologically, histologically, and transcriptionally recapitulates eosinophilic esophagitis. In vivo and in vitro, TGFβR1 variant–expressing epithelial cells are hyperproliferative, fail to differentiate properly, and overexpress innate proinflammatory mediators, which persist in the absence of lymphocytes or external allergens. Together, our results support the concept that TGFβ plays a fundamental, nonredundant, epithelial cell–intrinsic role in controlling tissue-specific allergic inflammation that is independent of its role in adaptive immunity. Eosinophilic esophagitis essentials: Individuals with loss-of-function variants of transforming growth factor–β receptor (TGFβR) genes develop allergic diseases including eosinophilic esophagitis (EoE), but mechanisms driving disease pathology are not well understood. Here Laky et al. define an essential role for TGFβR in maintaining epithelial cell homeostasis to control allergic inflammation. Mice bearing TGFβR1 loss-of-function variants developed disease symptoms consistent with EoE, which was reflected in pathological, immunological, and transcriptional changes to esophagus. Expression of TGFβR1 variants in epithelial cells was associated with hyperproliferation, defects in differentiation, and the release of pro-inflammatory mediators, even in the absence of allergens or lymphocytes. These findings highlight an epithelial cell–intrinsic function of TGFβR1 in modulating allergic inflammation associated with EoE. —CNF [ABSTRACT FROM AUTHOR]

Single-dose vaccines with the ability to restrict SARS-CoV-2 replication in the respiratory tract are needed for all age groups, aiding efforts toward control of COVID-19. We developed a live intranasal vector vaccine for infants and children against COVID-19 based on replication-competent chimeric bovine/human parainfluenza virus type 3 (B/HPIV3) that express the native (S) or prefusionstabilized (S-2P) SARS-CoV-2 S spike protein, the major protective and neutralization antigen of SARS-CoV-2. B/HPIV3/S and B/HPIV3/S-2P replicated as efficiently as B/HPIV3 in vitro and stably expressed SARS-CoV-2 S. Prefusion stabilization increased S expression by B/HPIV3 in vitro. In hamsters, a single intranasal dose of B/HPIV3/S-2P induced significantly higher titers compared to B/HPIV3/S of serum SARS-CoV-2–neutralizing antibodies (12- fold higher), serum IgA and IgG to SARS-CoV-2 S protein (5-fold and 13-fold), and IgG to the receptor binding domain (10-fold). Antibodies exhibited broad neutralizing activity against SARSCoV-2 of lineages A, B.1.1.7, and B.1.351. Four weeks after immunization, hamsters were challenged intranasally with 104.5 50% tissue-culture infectious-dose (TCID50) of SARS-CoV-2. In B/HPIV3 empty vector-immunized hamsters, SARS-CoV-2 replicated to mean titers of 106.6 TCID50/g in lungs and 107 TCID50/g in nasal tissues and induced moderate weight loss. In B/HPIV3/S-immunized hamsters, SARS-CoV-2 challenge virus was reduced 20-fold in nasal tissues and undetectable in lungs. In B/HPIV3/S-2P–immunized hamsters, infectious challenge virus was undetectable in nasal tissues and lungs; B/HPIV3/S and B/HPIV3/S-2P completely protected against weight loss after SARS-CoV-2 challenge. B/HPIV3/S-2P is a promising vaccine candidate to protect infants and young children against HPIV3 and SARS-CoV-2. [ABSTRACT FROM AUTHOR]

Rapid response: Protein subunit–based vaccines have been used extensively for protection against viral infections. Here, Francica et al. tested a protein subunit vaccine for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The authors vaccinated nonhuman primates with soluble prefusion-stabilized spike trimers (preS dTM) plus the adjuvant AS03, an oil-in-water emulsion. The authors found that preS dTM plus AS03 induced robust antibody and cellular immune responses that protected nonhuman primates from disease when challenged with SARS-CoV-2. This rapid protection, with increases in antibodies specific to spike protein observable as soon as 2 days after infection, provides evidence of a critical anamnestic antibody response. Antibodies elicited by preS dTM vaccination are protective against SARS-CoV-2 in nonhuman primates. Adjuvanted soluble protein vaccines have been used extensively in humans for protection against various viral infections based on their robust induction of antibody responses. Here, soluble prefusion-stabilized spike protein trimers (preS dTM) from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were formulated with the adjuvant AS03 and administered twice to nonhuman primates (NHPs). Binding and functional neutralization assays and systems serology revealed that the vaccinated NHP developed AS03-dependent multifunctional humoral responses that targeted distinct domains of the spike protein and bound to a variety of Fc receptors mediating immune cell effector functions in vitro. The neutralizing 50% inhibitory concentration titers for pseudovirus and live SARS-CoV-2 were higher than titers for a panel of human convalescent serum samples. NHPs were challenged intranasally and intratracheally with a high dose (3 × 106 plaque forming units) of SARS-CoV-2 (USA-WA1/2020 isolate). Two days after challenge, vaccinated NHPs showed rapid control of viral replication in both the upper and lower airways. Vaccinated NHPs also had increased spike protein–specific immunoglobulin G (IgG) antibody responses in the lung as early as 2 days after challenge. Moreover, passive transfer of vaccine-induced IgG to hamsters mediated protection from subsequent SARS-CoV-2 challenge. These data show that antibodies induced by the AS03-adjuvanted preS dTM vaccine were sufficient to mediate protection against SARS-CoV-2 in NHPs and that rapid anamnestic antibody responses in the lung may be a key mechanism for protection. [ABSTRACT FROM AUTHOR]

Released from RGS10 repression: RGS10 is a GTPase-activating protein (GAP) that inhibits signaling downstream of GPCRs, including those activated by chemokines. Chinn et al. characterized two RGS10 variants found in three brothers with growth impairment and recurrent infections. The authors found that increasing RGS10 abundance impaired the secretion of growth hormone from a pituitary adenoma cell line in response to the chemokine CXCL12, suggesting a potential mechanism for the growth hormone deficiency in these patients. They also found that the mutations did not affect the GAP activity of RGS10 but instead altered its PKA-mediated phosphorylation and subcellular localization, which they linked to the defective chemotaxis exhibited by patient-derived lymphocytes. Thus, this characterization of rare variants reveals that the repressive effects of RGS10 on GPCR signaling are critical for the proper function of the immune and endocrine systems. We report the clinical and molecular phenotype of three siblings from one family, who presented with short stature and immunodeficiency and carried uncharacterized variants in RGS10 (c.489_491del:p.E163del and c.G511T:p.A171S). This gene encodes regulator of G protein signaling 10 (RGS10), a member of a large family of GTPase-activating proteins (GAPs) that targets heterotrimeric G proteins to constrain the activity of G protein–coupled receptors, including receptors for chemoattractants. The affected individuals exhibited systemic abnormalities directly related to the RGS10 mutations, including recurrent infections, hypergammaglobulinemia, profoundly reduced lymphocyte chemotaxis, abnormal lymph node architecture, and short stature due to growth hormone deficiency. Although the GAP activity of each RGS10 variant was intact, each protein exhibited aberrant patterns of PKA-mediated phosphorylation and increased cytosolic and cell membrane localization and activity compared to the wild-type protein. We propose that the RGS10 p.E163del and p.A171S mutations lead to mislocalization of the RGS10 protein in the cytosol, thereby resulting in attenuated chemokine signaling. This study suggests that RGS10 is critical for both immune competence and normal hormonal metabolism in humans and that rare RGS10 variants may contribute to distinct systemic genetic disorders. [ABSTRACT FROM AUTHOR]

A rosy outlook for Roseomonas: In a clinical trial, topical application of the healthy skin bacterium Roseomonas mucosa improved atopic dermatitis (AD) in children age 3 years or older. R. mucosa treatment was associated with improvements in disease symptoms, epithelial barrier function, the amount of Staphylococcus aureus growing on the skin, the need for topical steroids, and quality of life for children and their families. Clinical improvements and colonization of skin by R. mucosa persisted for up to 8 months after treatment cessation and were not associated with major adverse events. Cell culture analyses and studies in the MC903 mouse model of AD suggest that the therapeutic mechanism may involve lipid production by R. mucosa, cholinergic signaling, and flagellin expression leading to a TNFR2-mediated epithelial-to-mesenchymal transition. Dysbiosis of the skin microbiota is increasingly implicated as a contributor to the pathogenesis of atopic dermatitis (AD). We previously reported first-in-human safety and clinical activity results from topical application of the commensal skin bacterium Roseomonas mucosa for the treatment of AD in 10 adults and 5 children older than 9 years of age. Here, we examined the potential mechanism of action of R. mucosa treatment and its impact on children with AD less than 7 years of age, the most common age group for children with AD. In 15 children with AD, R. mucosa treatment was associated with amelioration of disease severity, improvement in epithelial barrier function, reduced Staphylococcus aureus burden on the skin, and a reduction in topical steroid requirements without severe adverse events. Our observed response rates to R. mucosa treatment were greater than those seen in historical placebo control groups in prior AD studies. Skin improvements and colonization by R. mucosa persisted for up to 8 months after cessation of treatment. Analyses of cellular scratch assays and the MC903 mouse model of AD suggested that production of sphingolipids by R. mucosa, cholinergic signaling, and flagellin expression may have contributed to therapeutic impact through induction of a TNFR2-mediated epithelial-to-mesenchymal transition. These results suggest that a randomized, placebo-controlled trial of R. mucosa treatment in individuals with AD is warranted and implicate commensals in the maintenance of the skin epithelial barrier. [ABSTRACT FROM AUTHOR]

Transmission to chimpanzees of a precore hepatitis B virus (HBV) mutant implicated in acute liver failure (ALF) in humans did not cause ALF nor the classic form of acute hepatitis B (AHB) seen upon infection with the wild-type HBV strain, but rather a severe AHB with distinct disease features. Here, we investigated the viral and host immunity factors responsible for the unusual severity of AHB associated with the precore HBV mutant in chimpanzees. Archived serial serum and liver specimens from two chimpanzees inoculated with a precore HBV mutant implicated in ALF and two chimpanzees inoculated with wild-type HBV were studied. We used phage-display library and next-generation sequencing (NGS) technologies to characterize the liver antibody response. The results obtained in severe AHB were compared with those in classic AHB and HBV-associated ALF in humans. Severe AHB was characterized by: (i) the highest alanine aminotransferase (ALT) peaks ever seen in HBV transmission studies with a significantly shorter incubation period, compared to classic AHB; (ii) earlier HBsAg clearance and anti-HBs seroconversion with transient or undetectable hepatitis B e antigen (HBeAg); (iii) limited inflammatory reaction relative to hepatocellular damage at the ALT peak with B-cell infiltration, albeit less extensive than in ALF; (iv) detection of intrahepatic germline antibodies against hepatitis B core antigen (HBcAg) by phage-display libraries in the earliest disease phase, as seen in ALF; (v) lack of intrahepatic IgM anti-HBcAg Fab, as seen in classic AHB, but at variance with ALF; and (vi) higher proportion of antibodies in germline configuration detected by NGS in the intrahepatic antibody repertoire compared to classic AHB, but lower than in ALF. This study identifies distinct outcome-specific features associated with severe AHB caused by a precore HBV mutant in chimpanzees, which bear closer resemblance to HBV ALF than to classic AHB. Our data suggest that precore HBV mutants carry an inherently higher pathogenicity that, in addition to specific host factors, may play a critical role in determining the severity of acute HBV disease. Author summary: While the pathogenesis of classic acute hepatitis B (AHB) is believed to be mediated by hepatitis B virus (HBV)-specific T-cell responses, the pathogenesis of HBV-associated acute liver failure (ALF), one of the most rapid and lethal liver diseases, remains largely unknown. Our previous work demonstrated that ALF is associated with an uncommon B-cell response with the intrahepatic production of antibodies in germline configuration directed against the hepatitis B core antigen (HBcAg). Previous reports also documented an association of ALF with HBV variants containing precore or core promoter mutations. Here, we studied chimpanzees experimentally infected with a precore HBV mutant implicated in human ALF with the aim of getting new insights into ALF pathogenesis. Although these chimpanzees did not develop ALF, they had an unusually severe AHB with distinct disease features, including the highest alanine aminotransferase (ALT) peaks ever seen in HBV transmission studies, a shorter incubation period with an earlier ALT peak, an earlier HBsAg clearance and antibody seroconversion compared to classic AHB, and intrahepatic B-cell infiltration. Remarkably, the ALT peak correlated with extensive hepatocellular damage, but very limited inflammatory reaction. As previously documented in ALF, anti-HBcAg antibodies in germline configuration were present in the liver in the earliest disease phase. Thus, although chimpanzees did not develop ALF, infection with a precore HBV mutant caused a severe AHB with several analogies with ALF, suggesting that precore HBV mutants carry an inherently higher pathogenicity. [ABSTRACT FROM AUTHOR]

Leishmania donovani causes visceral leishmaniasis (VL), which is typically fatal without treatment. There is substantial variation between individuals in rates of disease progression, response to treatment and incidence of post-treatment sequelae, specifically post-kala-azar dermal leishmaniasis (PKDL). Nevertheless, the majority of infected people are asymptomatic carriers. Hamsters and mice are commonly used as models of fatal and non-fatal VL, respectively. Host and parasite genetics are likely to be important factors, but in general the reasons for heterogeneous disease presentation in humans and animal models are poorly understood. Host microbiota has become established as a factor in cutaneous forms of leishmaniasis but this has not been studied in VL. We induced intestinal dysbiosis in mice and hamsters by long-term treatment with broad-spectrum antibiotics in their drinking water. There were no significant differences in disease presentation in dysbiotic mice. In contrast, dysbiotic hamsters infected with L. donovani had delayed onset and progression of weight loss. Half of control hamsters had a rapid progression phenotype compared with none of the ABX-treated animals and the nine-month survival rate was significantly improved compared to untreated controls (40% vs. 10%). Antibiotic-treated hamsters also had significantly less severe hepatosplenomegaly, which was accompanied by a distinct cytokine gene expression profile. The protective effect was not explained by differences in parasite loads or haematological profiles. We further found evidence that the gut-liver axis is a key aspect of fatal VL progression in hamsters, including intestinal parasitism, bacterial translocation to the liver, malakoplakia and iron sequestration, none of which occurred in non-progressing murine VL. Diverse bacterial genera were cultured from VL affected livers, of which Rodentibacter was specifically absent from ABX-treated hamsters, indicating this pathobiont may play a role in promoting disease progression. The results provide experimental support for antibiotic prophylaxis against secondary bacterial infections as an adjunct therapy in human VL patients. Author summary: Visceral leishmaniasis (VL) is a potentially fatal neglected infectious disease that is widespread in South Asia, East Africa, South America and the Mediterranean region. VL is caused by infections with the protozoan parasite Leishmania donovani. Infected people may remain asymptomatic or progress, at variable rates, to develop VL disease affecting the liver, spleen and blood. The reasons why clinical outcomes vary so much is not well understood. We decided to investigate whether a host's gut microbiota might be a relevant factor. We treated mice and hamsters with antibiotics to disrupt their gut microbiota and then infected them with L. donovani. No effect was observed in mice, however, treated hamsters had slower onset and progression of VL, less severe enlargement of the liver and spleen, and had a reduced mortality rate. This was accompanied by alterations in the levels of key immune response factors. We also found novel aspects of symptomatic VL in hamsters relating to the gut and its microbiota. Specifically, the gut tissues themselves were parasitized by L. donovani and liver tissues became co-infected with both L. donovani and Gram negative bacteria originating from the gut. Overall, our findings support the inclusion of anti-bacterial therapy as part of VL treatment strategies. [ABSTRACT FROM AUTHOR]

Eosinophils are present in muscle lesions associated with Duchenne muscular dystrophy and dystrophin-deficient mdx mice that phenocopy this disorder. Although it has been hypothesized that eosinophils promote characteristic inflammatory muscle damage, this has not been fully examined. In this study, we generated mice with the dystrophin mutation introduced into PHIL, a strain with a transgene that directs lineage-specific eosinophil ablation. We also explored the impact of eosinophil overabundance on dystrophinopathy by introducing the dystrophin mutation into IL-5 transgenic mice. We evaluated the degree of eosinophil infiltration in association with myofiber size distribution, centralized nuclei, serum creatine kinase, and quantitative histopathology scores. Among our findings, eosinophils were prominent in the quadriceps muscles of 4-wk-old male mdx mice but no profound differences were observed in the quantitative measures of muscle damage when comparing mdx versus mdx.PHIL versus mdx.IL5tg mice, despite dramatic differences in eosinophil infiltration (CD45+CD11c-Gr1-MHC class IIloSiglecF+ eosinophils at 1.2 ± 0.34% versus <0.1% versus 20 ± 7.6% of total cells, respectively). Further evaluation revealed elevated levels of eosinophil chemoatttractants eotaxin-1 and RANTES in the muscle tissue of all three dystrophin-deficient strains; eotaxin-1 concentration in muscle correlated inversely with age. Cytokines IL-4 and IL-1R antagonist were also detected in association with eosinophils in muscle. Taken together, our findings challenge the long-held perception of eosinophils as cytotoxic in dystrophin-deficient muscle; we show clearly that eosinophil infiltration is not a driving force behind acute muscle damage in the mdx mouse strain. Ongoing studies will focus on the functional properties of eosinophils in this unique microenvironment. [ABSTRACT FROM AUTHOR]

Introduction: As therapies for atopic dermatitis (AD) based on live biotherapeutic products (LBP) are developed, the potential displacement of biotherapeutic strains, and species to mucosal sites where they are not naturally found is of investigative interest. However, formal assessment of the toxicity potential of healthy skin commensal organisms has not been reported in the literature. Our previous research indicates that topical application of live Roseomonas mucosa to treat AD was associated with clinical benefit on the skin, but the effects of exposure via inhalation, eye inoculation, and ingestion were unknown. Methods: Herein we report our findings from mice inoculated with commensal strains of R. mucosa , coagulase negative Staphylococci (CNS), and Pseudomonas aeruginosa. Bacterial isolates were collected under clinical trial NCT03018275, however these results do not represent an interventional clinical trial. Results: Our tested R. mucosa isolates did not display significant infection or inflammation. However, neutropenic mice inoculated with CNS had infection without major inflammation in pulmonary models. In contrast, systemic infection generated hepatic and splenic pathology for P. aeruginosa and CNS, which was worsened by the presence of neutropenia. Discussion: Our results suggest that LBP derived from bacteria without significant infectivity histories, such as R. mucosa , may represent safer options than known pathobionts like P. aeruginosa and Staphylococcus spp. Overall, these results suggest that topically applied LBP from select skin commensals are likely to present safe therapeutic options and reinforce our prior clinical findings. [ABSTRACT FROM AUTHOR]

Hepatitis B virus (HBV)-associated acute liver failure (ALF) is a dramatic clinical syndrome leading to death or liver transplantation in 80% of cases. Due to the extremely rapid clinical course, the difficulties in obtaining liver specimens, and the lack of an animal model, the pathogenesis of ALF remains largely unknown. Here, we performed a comprehensive genetic and functional characterization of the virus and the host in liver tissue from HBV-associated ALF and compared the results with those of classic acute hepatitis B in chimpanzees. In contrast with acute hepatitis B, HBV strains detected in ALF livers displayed highly mutated HBV core antigen (HBcAg), associated with increased HBcAg expression ex vivo, which was independent of viral replication levels. Combined gene and miRNA expression profiling revealed a dominant B cell disease signature, with extensive intrahepatic production of IgM and IgG in germline configuration exclusively targeting HBcAg with subnanomolar affinities, and complement deposition. Thus, HBV ALF appears to be an anomalous T cell-independent, HBV core-driven B cell disease, which results from the rare and unfortunate encounter between a host with an unusual B cell response and an infecting virus with a highly mutated core antigen. [ABSTRACT FROM AUTHOR]

Keratinocytes are the most abundant cell type in the epidermis. They prevent desiccation and provide immunological and barrier defense against potential pathogens such as Staphylococcus aureus and Candida albicans. The study of this first line of immune defense may be hindered by invasive isolation methods and/or improper culture conditions to support stem cell maintenance and other potential mechanisms contributing to long-term subcultivation in vitro. Primary keratinocytes have been successfully isolated from blister roofs induced by negative pressure, which separates the epidermis from the dermis in vivo in human subjects. This method allows collection of pure epidermal cells without dermal contamination in a minimally invasive manner. However, the isolated keratinocytes differentiate and senesce when cultured in vitro beyond five passages. Here, we present evidence that the Rho kinase (ROCK) inhibitor Y-27632 can be used to effectively increase the proliferative capabilities of keratinocytes isolated using the suction blister method, similar to what has been previously reported for primary keratinocytes isolated using alternative methods. We show that the increase in passage number is directly correlated to delayed differentiation, and that cells passaged long term with the inhibitor retain their ability to stratify in organotypic raft cultures and respond to cytokine treatment; additionally, the late passage cells have a heterogeneous mix of differentiated and non-differentiated cells which may be predicted by a ratio of select differentiation markers. The described method presents a minimally invasive procedure for keratinocyte isolation and prolonged culture that allows analysis of keratinocyte function in both healthy volunteers and patients with dermatologic diseases. [ABSTRACT FROM AUTHOR]

Salivary components from disease vectors help arthropods to acquire blood and have been shown to enhance pathogen transmission in different model systems. Here we show that two salivary enzymes from Lutzomyia longipalpis have a synergist effect that facilitates a more efficient blood meal intake and diffusion of other sialome components. We have previously shown that Lundep, a highly active endonuclease, enhances parasite infection and prevent blood clotting by inhibiting the intrinsic pathway of coagulation. To investigate the physiological role of a salivary hyaluronidase in blood feeding we cloned and expressed a recombinant hyaluronidase from Lu. longipalpis. Recombinant hyaluronidase (LuloHya) was expressed in mammalian cells and biochemically characterized in vitro. Our study showed that expression of neutrophil CXC chemokines and colony stimulating factors were upregulated in HMVEC cells after incubation with LuloHya and Lundep. These results were confirmed by the acute hemorrhage, edema and inflammation in a dermal necrosis (dermonecrotic) assay involving a massive infiltration of leukocytes, especially neutrophils, in mice co-injected with hemorrhagic factor and these two salivary proteins. Moreover, flow cytometry results showed that LuloHya and Lundep promote neutrophil recruitment to the bite site that may serve as a vehicle for establishment of Leishmania infection. A vaccination experiment demonstrated that LuloHya and Lundep confer protective immunity against cutaneous leishmaniasis using the Lu. longipalpis—Leishmania major combination as a model. Animals (C57BL/6) immunized with LuloHya or Lundep showed minimal skin damage while lesions in control animals remained ulcerated. This protective immunity was abrogated when B-cell-deficient mice were used indicating that antibodies against both proteins play a significant role for disease protection. Rabbit-raised anti-LuloHya antibodies completely abrogated hyaluronidase activity in vitro. Moreover, in vivo experiments demonstrated that blocking LuloHya with specific antibodies interferes with sand fly blood feeding. This work highlights the relevance of vector salivary components in blood feeding and parasite transmission and further suggests the inclusion of these salivary proteins as components for an anti-Leishmania vaccine. [ABSTRACT FROM AUTHOR]

Ticks transmit infectious agents including bacteria, viruses and protozoa. However, their transmission may be compromised by host resistance to repeated tick feeding. Increasing host resistance to repeated tick bites is well known in laboratory animals, including intense inflammation at the bite sites. However, it is not known whether this also occurs in wild rodents such as white-footed mice, Peromyscus leucopus, and other wildlife, or if it occurs at all. According to the "host immune incompetence" hypothesis, if these mice do not have a strong inflammatory response, they would not reject repeated tick bites by Ixodes scapularis. To test this hypothesis, histopathological studies were done comparing dermal inflammation in P. leucopus versus guinea pigs, Cavia porcellus, repeatedly infested with I. scapularis. In P. leucopus, the immune cell composition was like that seen in laboratory mouse models, with some differences. However, there was a broad sessile lesion with intact dermal architecture, likely enabling the ticks to continue feeding unimpeded. In contrast, in C. porcellus, there was a relatively similar mixed cellular profile, but there also was a large, leukocyte-filled cavitary lesion and scab-like hyperkeratotic changes to the epidermal layer, along with itching and apparent pain. Ticks attached to sensitized C. porcellus fed poorly or were dislodged, presumably due to the weakened anchoring of the tick's mouthparts cemented in the heavily inflamed and disintegrating dermal tissues. This is the first time that the architecture of the skin lesions has been recognized as a major factor in understanding tick-host tolerance versus tick bite rejection. These findings broadly strengthen previous work done on lab animal models but also help explain why I. scapularis can repeatedly parasitize whitefooted mice, supporting the "immune evasion theory" but cannot repeatedly parasitize other, non-permissive hosts such as guinea pigs. [ABSTRACT FROM AUTHOR]

The Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic betacoronavirus that was first detected in humans in 2012 as a cause of severe acute respiratory disease. As of July 28, 2017, there have been 2,040 confirmed cases with 712 reported deaths. While many infections have been fatal, there have also been a large number of mild or asymptomatic cases discovered through monitoring and contact tracing. New Zealand white rabbits are a possible model for asymptomatic infection with MERS-CoV. In order to discover more about non-lethal infections and to learn whether a single infection with MERS-CoV would protect against reinfection, we inoculated rabbits with MERS-CoV and monitored the antibody and inflammatory response. Following intranasal infection, rabbits developed a transient dose-dependent pulmonary infection with moderately high levels of viral RNA, viral antigen, and perivascular inflammation in multiple lung lobes that was not associated with clinical signs. The rabbits developed antibodies against viral proteins that lacked neutralizing activity and the animals were not protected from reinfection. In fact, reinfection resulted in enhanced pulmonary inflammation, without an associated increase in viral RNA titers. Interestingly, passive transfer of serum from previously infected rabbits to naïve rabbits was associated with enhanced inflammation upon infection. We further found this inflammation was accompanied by increased recruitment of complement proteins compared to primary infection. However, reinfection elicited neutralizing antibodies that protected rabbits from subsequent viral challenge. Our data from the rabbit model suggests that people exposed to MERS-CoV who fail to develop a neutralizing antibody response, or persons whose neutralizing antibody titers have waned, may be at risk for severe lung disease on re-exposure to MERS-CoV. [ABSTRACT FROM AUTHOR]

Ferrets are a valuable model for influenza virus pathogenesis, virus transmission, and antiviral therapy studies. However, the contributions of the volume of inoculum administered and the ferret's respiratory tract anatomy to disease outcome have not been explored. We noted variations in clinical disease outcomes and the volume of inoculum administered and investigated these differences by administering two influenza viruses (A/California/07/2009 [H1N1 pandemic] and A/Minnesota/11/2010 [H3N2 variant]) to ferrets intranasally at a dose of 106 50% tissue culture infective doses in a range of inoculum volumes (0.2, 0.5, or 1.0 ml) and followed viral replication, clinical disease, and pathology over 6 days. Clinical illness and respiratory tract pathology were the most severe and most consistent when the viruses were administered in a volume of 1.0 ml. Using a modified micro-computed tomography imaging method and examining gross specimens, we found that the right main-stem bronchus was consistently larger in diameter than the left main-stem bronchus, though the latter was longer and straighter. These anatomic features likely influence the distribution of the inoculum in the lower respiratory tract. A 1.0-ml volume of inoculum is optimal for delivery of virus to the lower respiratory tract of ferrets, particularly when evaluation of clinical disease is desired. Furthermore, we highlight important anatomical features of the ferret lung that influence the kinetics of viral replication, clinical disease severity, and lung pathology. [ABSTRACT FROM AUTHOR]

Chronic inhalation of fungi and fungal components has been linked to the development of respiratory disorders, although their role with respect to the pathogenesis of acute respiratory virus infection remains unclear. Here, we evaluate inflammatory pathology induced by repetitive administration of a filtrate of the ubiquitous fungus, Alternaria alternata, and its impact on susceptibility to infection with influenza A. We showed previously that A. alternata at the nasal mucosae resulted in increased susceptibility to an otherwise sublethal inoculum of influenza A in wild-type mice. Here we demonstrate that A. alternata-induced potentiation of influenza A infection was not dependent on fungal serine protease or ribonuclease activity. Repetitive challenge with A. alternata prior to virus infection resulted proinflammatory cytokines, neutrophil recruitment, and loss of alveolar macrophages to a degree that substantially exceeded that observed in response to influenza A infection alone. Concomitant administration of immunomodulatory Lactobacillus plantarum, a strategy shown previously to limit virus-induced inflammation in the airways, blocked the exaggerated lethal response. These observations promote an improved understanding of severe influenza infection with potential clinical relevance for individuals subjected to continuous exposure to molds and fungi. [ABSTRACT FROM AUTHOR]

Simian hemorrhagic fever virus (SHFV) causes a fulminant and typically lethal viral hemorrhagic fever (VHF) in macaques (Cercopithecinae: Macaca spp.) but causes subclinical infections in patas monkeys (Cercopithecinae: Erythrocebus patas). This difference in disease course offers a unique opportunity to compare host responses to infection by a VHF-causing virus in biologically similar susceptible and refractory animals. Patas and rhesus monkeys were inoculated side-by-side with SHFV. Unlike the severe disease observed in rhesus monkeys, patas monkeys developed a limited clinical disease characterized by changes in complete blood counts, serum chemistries, and development of lymphadenopathy. Viral RNA was measurable in circulating blood 2 days after exposure, and its duration varied by species. Infectious virus was detected in terminal tissues of both patas and rhesus monkeys. Varying degrees of overlap in changes in serum concentrations of interferon (IFN)-γ, monocyte chemoattractant protein (MCP)-1, and interleukin (IL)-6 were observed between patas and rhesus monkeys, suggesting the presence of common and species-specific cytokine responses to infection. Similarly, quantitative immunohistochemistry of livers from terminal monkeys and whole blood flow cytometry revealed varying degrees of overlap in changes in macrophages, natural killer cells, and T-cells. The unexpected degree of overlap in host response suggests that relatively small subsets of a host's response to infection may be responsible for driving hemorrhagic fever pathogenesis. Furthermore, comparative SHFV infection in patas and rhesus monkeys offers an experimental model to characterize host–response mechanisms associated with viral hemorrhagic fever and evaluate pan-viral hemorrhagic fever countermeasures. [ABSTRACT FROM AUTHOR]

Influenza viruses of the H1N1, H2N2, and H3N2 subtypes have caused previous pandemics. H2 influenza viruses represent a pandemic threat due to continued circulation in wild birds and limited immunity in the human population. In the event of a pandemic, antiviral agents are the mainstay for treatment, but broadly neutralizing antibodies (bNAbs) may be a viable alternative for short-term prophylaxis or treatment. The hemagglutinin stem binding bNAbs CR6261 and CR9114 have been shown to protect mice from severe disease following challenge with H1N1 and H5N1 and with H1N1, H3N2, and influenza B viruses, respectively. Early studies with CR6261 and CR9114 showed weak in vitro activity against human H2 influenza viruses, but the in vivo efficacy against H2 viruses is unknown. Therefore, we evaluated these antibodies against human- and animal-origin H2 viruses A/Ann Arbor/6/1960 (H2N2) (AA60) and A/swine/MO/4296424/06 (H2N3) (Sw06). In vitro, CR6261 neutralized both H2 viruses, while CR9114 only neutralized Sw06. To evaluate prophylactic efficacy, mice were given CR6261 or CR9114 and intranasally challenged 24 h later with lethal doses of AA60 or Sw06. Both antibodies reduced mortality, weight loss, airway inflammation, and pulmonary viral load. Using engineered bNAb variants, antibody-mediated cell cytotoxicity reporter assays, and Fcγ receptor-deficient (Fcer1g-/-) mice, we show that the in vivo efficacy of CR9114 against AA60 is mediated by Fcγ receptor-dependent mechanisms. Collectively, these findings demonstrate the in vivo efficacy of CR6261 and CR9114 against H2 viruses and emphasize the need for in vivo evaluation of bNAbs. IMPORTANCE bNAbs represent a strategy to prevent or treat infection by a wide range of influenza viruses. The evaluation of these antibodies against H2 viruses is important because H2 viruses caused a pandemic in 1957 and could cross into humans again. We demonstrate that CR6261 and CR9114 are effective against infection with H2 viruses of both human and animal origin in mice, despite the finding that CR9114 did not display in vitro neutralizing activity against the human H2 virus. These findings emphasize the importance of in vivo evaluation and testing of bNAbs. [ABSTRACT FROM AUTHOR]

Pediatric SARS-CoV-2 vaccines are needed that elicit immunity directly in the airways as well as systemically. Building on pediatric parainfluenza virus vaccines in clinical development, we generated a live-attenuated parainfluenza-virus-vectored vaccine candidate expressing SARS-CoV-2 prefusion-stabilized spike (S) protein (B/HPIV3/S-6P) and evaluated its immunogenicity and protective efficacy in rhesus macaques. A single intranasal/intratracheal dose of B/HPIV3/S-6P induced strong S-specific airway mucosal immunoglobulin A (IgA) and IgG responses. High levels of S-specific antibodies were also induced in serum, which efficiently neutralized SARS-CoV-2 variants of concern of alpha, beta, and delta lineages, while their ability to neutralize Omicron sub-lineages was lower. Furthermore, B/HPIV3/S-6P induced robust systemic and pulmonary S-specific CD4+ and CD8+ T cell responses, including tissue-resident memory cells in the lungs. Following challenge, SARS-CoV-2 replication was undetectable in airways and lung tissues of immunized macaques. B/HPIV3/S-6P will be evaluated clinically as pediatric intranasal SARS-CoV-2/parainfluenza virus type 3 vaccine. [Display omitted] • Single intranasal dose of B/HPIV3/S-6P protects macaques against SARS-CoV-2 challenge • B/HPIV3/S-6P induces strong mucosal and systemic IgM, IgA, and IgG responses to S, RBD • Intranasal immunization induces S-specific systemic and airway T cell responses • S-specific T cells in airways transition to tissue-resident memory phenotypes Use of an attenuated parainfluenza virus vector allows for effective vaccination against SARS-CoV-2 via the airways in young macaques, suggesting a promising approach for needle-free pediatric vaccination of humans against COVID-19. [ABSTRACT FROM AUTHOR]