CD151 Maintains Endolysosomal Protein Quality to Inhibit Vascular Inflammation.

BACKGROUND: Tetraspanin CD151 is highly expressed in endothelia and reinforces cell adhesion, but its role in vascular inflammation remains largely unknown. METHODS: In vitro molecular and cellular biological analyses on genetically modified endothelial cells, in vivo vascular biological analyses on genetically engineered mouse models, and in silico systems biology and bioinformatics analyses on CD151-related events. RESULTS: Endothelial ablation of Cd151 leads to pulmonary and cardiac inflammation, severe sepsis, and perilous COVID-19, and endothelial CD151 becomes downregulated in inflammation. Mechanistically, CD151 restrains endothelial release of proinflammatory molecules for less leukocyte infiltration. At the subcellular level, CD151 determines the integrity of multivesicular bodies/lysosomes and confines the production of exosomes that carry cytokines such as ANGPT2 (angiopoietin-2) and proteases such as cathepsin-D. At the molecular level, CD151 docks VCP (valosin-containing protein)/p97, which controls protein quality via mediating deubiquitination for proteolytic degradation, onto endolysosomes to facilitate VCP/p97 function. At the endolysosome membrane, CD151 links VCP/p97 to (1) IFITM3 (interferon-induced transmembrane protein 3), which regulates multivesicular body functions, to restrain IFITM3-mediated exosomal sorting, and (2) V-ATPase, which dictates endolysosome pH, to support functional assembly of V-ATPase. CONCLUSIONS: Distinct from its canonical function in strengthening cell adhesion at cell surface, CD151 maintains endolysosome function by sustaining VCP/p97-mediated protein unfolding and turnover. By supporting protein quality control and protein degradation, CD151 prevents proteins from (1) buildup in endolysosomes and (2) discharge through exosomes, to limit vascular inflammation. Also, our study conceptualizes that balance between degradation and discharge of proteins in endothelial cells determines vascular information. Thus, the IFITM3/V-ATPase-tetraspanin-VCP/p97 complexes on endolysosome, as a protein quality control and inflammation-inhibitory machinery, could be beneficial for therapeutic intervention against vascular inflammation.

Early and mid-gestation Zika virus (ZIKV) infection in the olive baboon (Papio anubis) leads to fetal CNS pathology by term gestation.

Zika virus (ZIKV) infection in pregnancy can produce catastrophic teratogenic damage to the developing fetus including microcephaly and congenital Zika syndrome (CZS). We previously described fetal CNS pathology occurring by three weeks post-ZIKV inoculation in Olive baboons at mid-gestation, including neuroinflammation, loss of radial glia (RG), RG fibers, neuroprogenitor cells (NPCs) resulting in disrupted NPC migration. In the present study, we explored fetal brain pathologies at term gestation resulting from ZIKV exposure during either first or second trimester in the Olive baboon. In all dams, vRNA in whole blood resolved after 7 days post inoculation (dpi). One first trimester infected dam aborted at 5 dpi. All dams developed IgM and IgG response to ZIKV with ZIKV IgG detected in fetal serum. Placental pathology and inflammation were observed including disruption of syncytiotrophoblast layers, delayed villous maturation, partially or fully thrombosed vessels, calcium mineralization and fibrin deposits. In the uterus, ZIKV was detected in ¾ first trimester but not in second trimester infected dams. While ZIKV was not detected in any fetal tissue at term, all fetuses exhibited varying degrees of neuropathology. Fetal brains from ZIKV inoculated dams exhibited a range of gross brain pathologies including irregularities of the major gyri and sulci of the cerebral cortex and cerebellar pathology. Frontal cortices of ZIKV fetuses showed a general disorganization of the six-layered cortex with degree of disorganization varying among the fetuses from the two groups. Frontal cortices from ZIKV inoculation in the first but not second trimester exhibited increased microglia, and in both trimester ZIKV inoculation, increased astrocyte numbers (white matter). In the cerebellum, increased microglia were observed in fetuses from both first and second trimester inoculation. In first trimester ZIKV inoculation, decreased oligodendrocyte precursor cell populations were observed in fetal cerebellar white matter. In general, our observations are in accordance with those described in human ZIKV infected fetuses.

Antigen Discovery for Next-Generation Pertussis Vaccines Using Immunoproteomics and Transposon-Directed Insertion Sequencing.

BACKGROUND: Despite high vaccination rates, the United States has experienced a resurgence in reported cases of pertussis after switching to the acellular pertussis vaccine, indicating a need for improved vaccines that enhance infection control. METHODS: Bordetella pertussis antigens recognized by convalescent-baboon serum and nasopharyngeal wash were identified by immunoproteomics and their subcellular localization predicted. Genes essential or important for persistence in the baboon airway were identified by transposon-directed insertion-site sequencing (TraDIS) analysis. RESULTS: In total, 314 B. pertussis antigens were identified by convalescent baboon serum and 748 by nasopharyngeal wash. Thirteen antigens were identified as immunogenic in baboons, essential for persistence in the airway by TraDIS, and membrane-localized: BP0840 (OmpP), Pal, OmpA2, BP1485, BamA, Pcp, MlaA, YfgL, BP2197, BP1569, MlaD, ComL, and BP0183. CONCLUSIONS: The B. pertussis antigens identified as immunogenic, essential for persistence in the airway, and membrane-localized warrant further investigation for inclusion in vaccines designed to reduce or prevent carriage of bacteria in the airway of vaccinated individuals.

Maternal Zika Virus (ZIKV) Infection following Vaginal Inoculation with ZIKV-Infected Semen in Timed-Pregnant Olive Baboons.

Zika virus (ZIKV) infection is now firmly linked to congenital Zika syndrome (CZS), including fetal microcephaly. While Aedes species of mosquito are the primary vector for ZIKV, sexual transmission of ZIKV is a significant route of infection. ZIKV has been documented in human, mouse, and nonhuman primate (NHP) semen. It is critical to establish NHP models of the vertical transfer of ZIKV that recapitulate human pathogenesis. We hypothesized that vaginal deposition of ZIKV-infected baboon semen would lead to maternal infection and vertical transfer in the olive baboon (Papio anubis). Epidemiological studies suggest an increased rate of CZS in the Americas compared to the original link to CZS in French Polynesia; therefore, we also compared the French Polynesian (FP) ZIKV isolate to the Puerto Rican (PR) isolate. Timed-pregnant baboons (n = 6) were inoculated via vaginal deposition of baboon semen containing 106 focus-forming units (FFU) of ZIKV (n = 3 for FP isolate H/PF/2013; n = 3 for PR isolate PRVABC59) at midgestation (86 to 95 days of gestation [dG]; term, 183 dG) on day 0 (all dams) and then at 7-day intervals through 3 weeks. Maternal blood, saliva, and cervicovaginal wash (CVW) samples were obtained. Animals were euthanized at 28 days (n = 5) or 39 days (n = 1) after the initial inoculation, and maternal/fetal tissues were collected. Viremia was achieved in 3/3 FP ZIKV-infected dams and 2/3 PR ZIKV-infected dams. ZIKV RNA was detected in CVW samples of 5/6 dams. ZIKV RNA was detected in lymph nodes but not the ovaries, uterus, cervix, or vagina in FP isolate-infected dams. ZIKV RNA was detected in lymph nodes (3/3), uterus (2/3), and vagina (2/3) in PR isolate-infected dams. Placenta, amniotic fluid, and fetal tissues were ZIKV RNA negative in the FP isolate-infected dams, whereas 2/3 PR isolate-infected dam placentas were ZIKV RNA positive. We conclude that ZIKV-infected semen is a means of ZIKV transmission during pregnancy in primates. The PR isolate appeared more capable of widespread dissemination to tissues, including reproductive tissues and placenta, than the FP isolate.IMPORTANCE Zika virus remains a worldwide health threat, with outbreaks still occurring in the Americas. While mosquitos are the primary vector for the spread of the virus, sexual transmission of Zika virus is also a significant means of infection, especially in terms of passage from an infected to an uninfected partner. While sexual transmission has been documented in humans, and male-to-female transmission has been reported in mice, ours is the first study in nonhuman primates to demonstrate infection via vaginal deposition of Zika virus-infected semen. The latter is important since a recent publication indicated that human semen inhibited, in a laboratory setting, Zika virus infection of reproductive tissues. We also found that compared to the French Polynesian isolate, the Puerto Rican Zika virus isolate led to greater spread throughout the body, particularly in reproductive tissues. The American isolates of Zika virus appear to have acquired mutations that increase their efficacy.

Zika virus infection at mid-gestation results in fetal cerebral cortical injury and fetal death in the olive baboon.

Zika virus (ZIKV) infection during pregnancy in humans is associated with an increased incidence of congenital anomalies including microcephaly as well as fetal death and miscarriage and collectively has been referred to as Congenital Zika Syndrome (CZS). Animal models for ZIKV infection in pregnancy have been developed including mice and non-human primates (NHPs). In macaques, fetal CZS outcomes from maternal ZIKV infection range from none to significant. In the present study we develop the olive baboon (Papio anubis), as a model for vertical transfer of ZIKV during pregnancy. Four mid-gestation, timed-pregnant baboons were inoculated with the French Polynesian ZIKV isolate (104 ffu). This study specifically focused on the acute phase of vertical transfer. Dams were terminated at 7 days post infection (dpi; n = 1), 14 dpi (n = 2) and 21 dpi (n = 1). All dams exhibited mild to moderate rash and conjunctivitis. Viremia peaked at 5-7 dpi with only one of three dams remaining mildly viremic at 14 dpi. An anti-ZIKV IgM response was observed by 14 dpi in all three dams studied to this stage, and two dams developed a neutralizing IgG response by either 14 dpi or 21 dpi, the latter included transfer of the IgG to the fetus (cord blood). A systemic inflammatory response (increased IL2, IL6, IL7, IL15, IL16) was observed in three of four dams. Vertical transfer of ZIKV to the placenta was observed in three pregnancies (n = 2 at 14 dpi and n = 1 at 21 dpi) and ZIKV was detected in fetal tissues in two pregnancies: one associated with fetal death at ~14 dpi, and the other in a viable fetus at 21 dpi. ZIKV RNA was detected in the fetal cerebral cortex and other tissues of both of these fetuses. In the fetus studied at 21 dpi with vertical transfer of virus to the CNS, the frontal cerebral cortex exhibited notable defects in radial glia, radial glial fibers, disorganized migration of immature neurons to the cortical layers, and signs of pathology in immature oligodendrocytes. In addition, indices of pronounced neuroinflammation were observed including astrogliosis, increased microglia and IL6 expression. Of interest, in one fetus examined at 14 dpi without detection of ZIKV RNA in brain and other fetal tissues, increased neuroinflammation (IL6 and microglia) was observed in the cortex. Although the placenta of the 14 dpi dam with fetal death showed considerable pathology, only minor pathology was noted in the other three placentas. ZIKV was detected immunohistochemically in two placentas (14 dpi) and one placenta at 21 dpi but not at 7 dpi. This is the first study to examine the early events of vertical transfer of ZIKV in a NHP infected at mid-gestation. The baboon thus represents an additional NHP as a model for ZIKV induced brain pathologies to contrast and compare to humans as well as other NHPs.

Local immune responses to tuberculin skin challenge in Mycobacterium bovis BCG-vaccinated baboons: a pilot study of younger and older animals.

Individuals over the age of 65 are highly susceptible to infectious diseases, which account for one-third of deaths in this age group. Vaccines are a primary tool to combat infection, yet they are less effective in the elderly population. While many groups have aimed to address this problem by studying vaccine-induced peripheral blood responses in the elderly, work from our lab and others demonstrate that immune responses to vaccination and infectious challenge may differ between tissue sites and the periphery. In this pilot study, we established an in vivo delayed-type hypersensitivity model of Mycobacterium bovis BCG vaccination and tuberculin skin test in two adult and two aged baboons. Vaccination generates BCG-specific immune cells that are recruited to the skin upon tuberculin challenge. We tested short term recall responses (8 weeks post-vaccination) and long term recall responses (25 weeks post-vaccination) by performing skin punch biopsies around the site of tuberculin injection. In short term recall responses, we found increased oxidation and decreased production of immune proteins in aged baboon skin at the site of TST challenge, in comparison to adult skin. Differences between adult and aged animals normalized in the long term response to tuberculin. In vitro, aged peripheral blood mononuclear cells had increased migration and functional responses to antigen-specific stimulation, suggesting that age-related changes in the tissue in vivo impairs aged immune recall responses to antigenic challenge. These findings highlight the impact of age-associated changes in the local tissue environment in memory recall responses, which may be more broadly applied to the study of other tissues. Moreover, these findings should be considered in future studies aimed at understanding and improving aging immune responses to vaccination and tissue challenge.

Zika Virus Infection, Reproductive Organ Targeting, and Semen Transmission in the Male Olive Baboon.

Zika virus (ZIKV) infection in pregnant women is a serious threat to the development and viability of the fetus. The primary mode of ZIKV transmission to humans is through mosquito bites, but sexual transmission has also been well documented in humans. However, little is known of the short- and long-term effects of ZIKV infection on the human male reproductive system. This study examines the effects of ZIKV infection on the male reproductive organs and semen and the immune response of the olive baboon (Papio anubis). Nine mature male baboons were infected with ZIKV (French Polynesian strain) subcutaneously. Six animals were euthanized at 41 days, while three animals were euthanized at 10 or 11 days postinfection (dpi). Viremia and clinical evidence of infection were present in all nine baboons. ZIKV RNA was present in the semen of five of nine baboons. ZIKV was present in the testes of two of three males euthanized at 10 or 11 dpi, but in none of six males at 41 dpi. Immunofluorescence of testes suggested the presence of ZIKV in sperm progenitor cells, macrophage penetration of seminiferous tubules, and increased tumor necrosis factor alpha (TNF-α), particularly in vascular walls. These data demonstrate that male olive baboons approximate the male human ZIKV response, including viremia, the adaptive immune response, and persistent ZIKV in semen. Although gross testicular pathology was not seen, the demonstrated breach of the testes-blood barrier and targeting of spermatogenic precursors suggest possible long-term implications in ZIKV-infected primates.IMPORTANCE Zika virus (ZIKV) is an emerging flavivirus spread through mosquitoes and sexual contact. ZIKV infection during pregnancy can lead to severe fetal outcomes, including miscarriage, fetal death, preterm birth, intrauterine growth restriction, and fetal microcephaly, collectively known as congenital Zika syndrome. Therefore, it is important to understand how this virus spreads, as well as the resulting pathogenesis in translational animal models that faithfully mimic ZIKV infection in humans. Such models will contribute to the future development of efficient therapeutics and prevention mechanisms. Through our previous work in olive baboons, we developed a nonhuman primate model that is permissive to ZIKV infection and transfers the virus vertically from mother to fetus, modeling human observations. The present study contributes to understanding of ZIKV infection in male baboon reproductive tissues and begins to elucidate how this may affect fertility, reproductive capacity, and sexual transmission of the virus.

Translational Model of Zika Virus Disease in Baboons.

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus with devastating outcomes seen recently in the Americas due to the association of maternal ZIKV infection with fetal microcephaly and other fetal malformations not previously associated with flavivirus infections. Here, we have developed the olive baboon (Papio anubis) as a nonhuman primate (NHP) translational model for the study of ZIKV pathogenesis and associated disease outcomes to contrast and compare with humans and other major NHPs, such as macaques. Following subcutaneous inoculation of adult male and nonpregnant female baboons, viremia was detected at 3 and 4 days postinfection (dpi) with the concordant presentation of a visible rash and conjunctivitis, similar to human ZIKV infection. Furthermore, virus was detected in the mucosa and cerebrospinal fluid. A robust ZIKV-specific IgM and IgG antibody response was also observed in all the animals. These data show striking similarity between humans and the olive baboon following infection with ZIKV, suggesting our model is a suitable translational NHP model to study ZIKV pathogenesis and potential therapeutics.IMPORTANCE ZIKV was first identified in 1947 in a sentinel rhesus monkey in Uganda and subsequently spread to Southeast Asia. Until 2007, only a small number of cases were reported, and ZIKV infection was relatively minor until the South Pacific and Brazilian outbreaks, where more severe outcomes were reported. Here, we present the baboon as a nonhuman primate model for contrast and comparison with other published animal models of ZIKV, such as the mouse and macaque species. Baboons breed year round and are not currently a primary nonhuman primate species used in biomedical research, making them more readily available for studies other than human immunodeficiency virus studies, which many macaque species are designated for. This, taken together with the similarities baboons have with humans, such as immunology, reproduction, genetics, and size, makes the baboon an attractive NHP model for ZIKV studies in comparison to other nonhuman primates.

Maternal Vaccination With a Monocomponent Pertussis Toxoid Vaccine Is Sufficient to Protect Infants in a Baboon Model of Whooping Cough.

Background: Bordetella pertussis is a human pathogen responsible for serious respiratory illness. The disease is most severe in infants too young to be vaccinated with most hospitalizations and deaths occurring within this age group. The Advisory Committee on Immunization Practices recommended immunization of pregnant women to protect infants from birth until their first vaccination at 6-8 weeks of age. We previously demonstrated that maternal vaccination with licensed acellular pertussis vaccines protected newborn baboons from disease. We hypothesized that protection was due to toxin-neutralizing, maternal anti-pertussis toxin antibodies and predicted that maternal vaccination with a pertussis toxoid (PTx)-only vaccine would protect newborns from disease. Methods: Infant baboons born to unvaccinated mothers or mothers vaccinated with a PTx-only vaccine were challenged with B. pertussis at 5 weeks of age and followed for infection and signs of disease. Results: Although all challenged infants were heavily colonized, the infant baboons born to mothers vaccinated with PTx-only vaccine were free from clinical disease following exposure to B. pertussis. In contrast, disease was observed in infants born to unvaccinated mothers. Conclusions: Our results demonstrated that maternal vaccination with a PTx-only vaccine is sufficient to protect newborn baboons from disease following exposure to pertussis.

Histopathology of Bordetella pertussis in the Baboon Model.

Pertussis is a severe respiratory disease caused by Bordetella pertussis The classic symptoms of pertussis include paroxysmal coughing with an inspiratory whoop, posttussive vomiting, cyanosis, and persistent coryzal symptoms. Infants under 2 months of age experience more severe disease, with most deaths occurring in this age group. Most of what is known about the pathology of pertussis in humans is from the evaluation of fatal human infant cases. The baboon model of pertussis provides the opportunity to evaluate the histopathology of severe but nonfatal pertussis. The baboon model recapitulates the characteristic clinical signs of pertussis observed in humans, including leukocytosis, paroxysmal coughing, mucus production, heavy colonization of the airway, and transmission of the bacteria between hosts. As in humans, baboons demonstrate age-related differences in clinical presentation, with younger animals experiencing more severe disease. We examined the histopathology of 5- to 6-week-old baboons, with the findings being similar to those reported for fatal human infant cases. In juvenile baboons, we found that the disease is highly inflammatory and concentrated to the lungs with signs of disease that would typically be diagnosed as acute respiratory distress syndrome (ARDS) and bronchopneumonia. In contrast, no significant pathology was observed in the trachea. Histopathological changes in the trachea were limited to cellular infiltrates and mucus production. Immunohistostaining revealed that the bacteria were localized to the surface of the ciliated epithelium in the conducting airways. Our observations provide important insights into the pathology of pertussis in typical, severe but nonfatal pertussis cases in a very relevant animal model.

Apnea induction for invasive lung function testing in infant olive baboons: Comparison of intravenous propofol versus hyperventilation.

BACKGROUND: In various types of pulmonary research, pulmonary function testing (PFT) is performed to quantify the severity of lung disease. Induction of apnea and positive pressure ventilation are required for accurate PFT measurements in non-cooperative subjects. We compared two methods of apnea induction in infant olive baboons (Papio anubis). METHODS: Pulmonary function testing results were compared during apnea induced by hyperventilation (CO2 washout) vs. intravenous propofol (1 dose 10 mg/kg). PFT was evaluated using a hot-wire pneumotachometer incorporated within an Avea ventilator in nine 1-month-old baboons. RESULTS: Propofol induced apnea faster and more reliably. In both groups, PFT values passed the statistical equivalence test and were not significantly different (Student's t-test). There was a trend toward less data variability after propofol administration. CONCLUSIONS: Intravenous propofol was non-inferior to CO2 washout for apnea induction in infant olive baboons. Propofol induced apnea faster and more reliably and yielded less variable PFT results.

Live Attenuated Pertussis Vaccine BPZE1 Protects Baboons Against Bordetella pertussis Disease and Infection.

Evidence suggests that the resurgence of pertussis in many industrialized countries may result from the failure of current vaccines to prevent nasopharyngeal colonization by Bordetella pertussis, the principal causative agent of whooping cough. Here, we used a baboon model to test the protective potential of the novel, live attenuated pertussis vaccine candidate BPZE1. A single intranasal/intratracheal inoculation of juvenile baboons with BPZE1 resulted in transient nasopharyngeal colonization and induction of immunoglobulin G and immunoglobulin A to all antigens tested, while causing no adverse symptoms or leukocytosis. When BPZE1-vaccinated baboons were challenged with a high dose of a highly virulent B. pertussis isolate, they were fully protected against disease, whereas naive baboons developed illness (with 1 death) and leukocytosis. Total postchallenge nasopharyngeal virulent bacterial burden of vaccinated animals was substantially reduced (0.002%) compared to naive controls, providing promising evidence in nonhuman primates that BPZE1 protects against both pertussis disease and B. pertussis infection.

Maternal and neonatal vaccination protects newborn baboons from pertussis infection.

BACKGROUND: The United States is experiencing a pertussis resurgence that resulted in a 60-year high of 48 000 cases in 2012. The majority of hospitalizations and deaths occur in infants too young to be vaccinated. Neonatal and maternal vaccination have been proposed to protect newborns until the first vaccination, currently recommended at 2 months of age. These interventions result in elevated anti-Bordetella pertussis titers, but there have been no studies demonstrating that these measures confer protection. METHODS: Baboons were vaccinated with acellular pertussis vaccine at 2 days of age or at 2 and 28 days of age. To model maternal vaccination, adult female baboons primed with acellular pertussis vaccine were boosted in the third trimester of pregnancy. Neonatally vaccinated infants, infants born to vaccinated mothers, and naive infants born to unvaccinated mothers were infected with B. pertussis at 5 weeks of age. RESULTS: Naive infant baboons developed severe disease when challenged with B. pertussis at 5 weeks of age. Baboons receiving acellular pertussis vaccine and infants born to mothers vaccinated at the beginning of their third trimester were protected. CONCLUSIONS: Our results demonstrate that neonatal vaccination and maternal vaccination confer protection in the baboon model and support further study of these strategies for protection of newborns from pertussis.

Recombinant Rift Valley fever vaccines induce protective levels of antibody in baboons and resistance to lethal challenge in mice.

Rift Valley fever (RVF) is a zoonotic disease endemic in Africa and the Arabian Peninsula caused by the highly infectious Rift Valley fever virus (RVFV) that can be lethal to humans and animals and results in major losses in the livestock industry. RVF is exotic to the United States; however, mosquito species native to this region can serve as biological vectors for the virus. Thus, accidental or malicious introduction of this virus could result in RVFV becoming endemic in North America. Such an event would likely lead to significant morbidity and mortality in humans, and devastating economic effects on the livestock industry. Currently, there are no licensed vaccines for RVF that are both safe and efficacious. To address this issue, we developed two recombinant RVFV vaccines using vaccinia virus (VACV) as a vector for use in livestock. The first vaccine, vCOGnGc, was attenuated by the deletion of a VACV gene encoding an IFN-γ binding protein, insertional inactivation of the thymidine kinase gene, and expression of RVFV glycoproteins, Gn and Gc. The second vaccine, vCOGnGcγ, is identical to the first and also expresses the human IFN-γ gene to enhance safety. Both vaccines are extremely safe; neither resulted in weight loss nor death in severe combined immunodeficient mice, and pock lesions were smaller in baboons compared with the controls. Furthermore, both vaccines induced protective levels of antibody titers in vaccinated mice and baboons. Mice were protected from lethal RVFV challenge. Thus, we have developed two safe and efficacious recombinant vaccines for RVF.

N-dihydrogalactochitosan serves as an effective mucosal adjuvant for intranasal vaccine in combination with recombinant viral proteins against respiratory infection.

Mucosal vaccinations for respiratory pathogens provide effective protection as they stimulate localized cellular and humoral immunities at the site of infection. Currently, the major limitation of intranasal vaccination is using effective adjuvants capable of withstanding the harsh environment imposed by the mucosa. Herein, we describe the efficacy of using a unique biopolymer, N-dihydrogalactochitosan (GC), as a nasal mucosal vaccine adjuvant against respiratory infections. Specifically, we mixed GC with recombinant SARS-CoV-2 trimeric spike (S) and nucleocapsid (NC) proteins to intranasally vaccinate K18-hACE2 transgenic mice, in comparison with Addavax (AV), an MF-59 equivalent. In contrast to AV, intranasal application of GC induces a robust, systemic antigen-specific antibody response and increases the number of T cells in the cervical lymph nodes. Moreover, GC+S+NC-vaccinated animals were largely resistant to the lethal SARS-CoV-2 challenge and experienced drastically reduced morbidity and mortality, with animal weights and behavior returning to normal 22 days post-infection. In contrast, animals intranasally vaccinated with AV+S+NC experienced severe weight loss, mortality, and respiratory distress, with none surviving beyond 6 days post-infection. Our findings demonstrate that GC can serve as a potent mucosal vaccine adjuvant against SARS-CoV-2 and potentially other respiratory viruses. STATEMENT OF SIGNIFICANCE: We demonstrated that a unique biopolymer, N-dihydrogalactochitosan (GC), was an effective nasal mucosal vaccine adjuvant against respiratory infections. Specifically, we mixed GC with recombinant SARS-CoV-2 trimeric spike (S) and nucleocapsid (NC) proteins to intranasally vaccinate K18-hACE2 transgenic mice, in comparison with Addavax (AV). In contrast to AV, GC induces a robust, systemic antigen-specific antibody response and increases the number of T cells in the cervical lymph nodes. About 90 % of the GC+S+NC-vaccinated animals survived the lethal SARS-CoV-2 challenge and remained healthy 22 days post-infection, while the AV+S+NC-vaccinated animals experienced severe weight loss and respiratory distress, and all died within 6 days post-infection. Our findings demonstrate that GC is a potent mucosal vaccine adjuvant against SARS-CoV-2 and potentially other respiratory viruses.

Infant baboons infected with respiratory syncytial virus develop clinical and pathological changes that parallel those of human infants.

Respiratory syncytial virus (RSV) infection of the lower respiratory tract is the leading cause of respiratory failure among infants in the United States of America and annually results in >300,000 deaths worldwide. Despite the importance of RSV, there is no licensed vaccine, and no specific form of therapy. This is largely due to the absence of an appropriate animal model for the evaluation of vaccines and therapeutic agents. We inoculated anesthetized infant (4 wk) baboons (Papio anubis) with a human strain of RSV intranasally or intratracheally. Baboons were monitored daily for clinical changes. Anesthetized baboons were intubated at various intervals, and bronchoalveolar lavage (BAL) was performed for viral culture and determination of leukocyte counts. Sham-infected baboons served as controls. Necropsies were performed on infected baboons on days 1, 3, 5, 8, or 13 after inoculation, with pathological analysis and immunohistochemical staining of lung tissues to detect RSV antigen. Infected baboons developed tachypnea and reduced oxygenation peaking from 4 to 8 days after infection and persisting for ≥14 days. Virus was recoverable in BAL fluid up to 8 days following infection. Necropsy revealed intense interstitial pneumonia, sloughing of the bronchiolar epithelium, and obstruction of the bronchiolar lumen with inflammatory cells and sloughed epithelial cells. RSV antigen was identified in bronchiolar and alveolar epithelium. We conclude that RSV-infected infant baboons develop clinical and pathological changes that parallel those observed in human infants with RSV infection. The infant baboon represents a much-needed model for studying the pathogenesis of RSV infection and evaluating antivirals and vaccines.

The Isolation and In Vitro Differentiation of Primary Fetal Baboon Tracheal Epithelial Cells for the Study of SARS-CoV-2 Host-Virus Interactions.

The mucociliary airway epithelium lines the human airways and is the primary site of host-environmental interactions in the lung. Following virus infection, airway epithelial cells initiate an innate immune response to suppress virus replication. Therefore, defining the virus-host interactions of the mucociliary airway epithelium is critical for understanding the mechanisms that regulate virus infection, including Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Non-human primates (NHP) are closely related to humans and provide a model to study human disease. However, ethical considerations and high costs can restrict the use of in vivo NHP models. Therefore, there is a need to develop in vitro NHP models of human respiratory virus infection that would allow for rapidly characterizing virus tropism and the suitability of specific NHP species to model human infection. Using the olive baboon (Papio anubis), we have developed methodologies for the isolation, in vitro expansion, cryopreservation, and mucociliary differentiation of primary fetal baboon tracheal epithelial cells (FBTECs). Furthermore, we demonstrate that in vitro differentiated FBTECs are permissive to SARS-CoV-2 infection and produce a potent host innate-immune response. In summary, we have developed an in vitro NHP model that provides a platform for the study of SARS-CoV-2 infection and other human respiratory viruses.

Single-cell transcriptomics reveals that tumor-infiltrating natural killer cells are activated by localized ablative immunotherapy and share anti-tumor signatures induced by immune checkpoint inhibitors.

Rationale: Natural killer (NK) cells provide protective anti-cancer immunity. However, the cancer therapy induced activation gene signatures and pathways in NK cells remain unclear. Methods: We applied a novel localized ablative immunotherapy (LAIT) by synergizing photothermal therapy (PTT) with intra-tumor delivering of the immunostimulant N-dihydrogalactochitosan (GC), to treat breast cancer using a mammary tumor virus-polyoma middle tumor-antigen (MMTV-PyMT) mouse model. We performed single-cell RNA sequencing (scRNAseq) analysis to unveil the cellular heterogeneity and compare the transcriptional alterations induced by PTT, GC, and LAIT in NK cells within the tumor microenvironment (TME). Results: ScRNAseq showed that NK subtypes, including cycling, activated, interferon-stimulated, and cytotoxic NK cells. Trajectory analysis revealed a route toward activation and cytotoxicity following pseudotime progression. Both GC and LAIT elevated gene expression associated with NK cell activation, cytolytic effectors, activating receptors, IFN pathway components, and cytokines/chemokines in NK subtypes. Single-cell transcriptomics analysis using immune checkpoint inhibitor (ICI)-treated animal and human samples revealed that ICI-induced NK activation and cytotoxicity across several cancer types. Furthermore, ICI-induced NK gene signatures were also induced by LAIT treatment. We also discovered that several types of cancer patients had significantly longer overall survival when they had higher expression of genes in NK cells that were also specifically upregulated by LAIT. Conclusion: Our findings show for the first time that LAIT activates cytotoxicity in NK cells and the upregulated genes positively correlate with beneficial clinical outcomes for cancer patients. More importantly, our results further establish the correlation between the effects of LAIT and ICI on NK cells, hence expanding our understanding of mechanism of LAIT in remodeling TME and shedding light on the potentials of NK cell activation and anti-tumor cytotoxic functions in clinical applications.

Western diet-induced shifts in the maternal microbiome are associated with altered microRNA expression in baboon placenta and fetal liver.

Maternal consumption of a high-fat, Western-style diet (WD) disrupts the maternal/infant microbiome and contributes to developmental programming of the immune system and nonalcoholic fatty liver disease (NAFLD) in the offspring. Epigenetic changes, including non-coding miRNAs in the fetus and/or placenta may also underlie this risk. We previously showed that obese nonhuman primates fed a WD during pregnancy results in the loss of beneficial maternal gut microbes and dysregulation of cellular metabolism and mitochondrial dysfunction in the fetal liver, leading to a perturbed postnatal immune response with accelerated NAFLD in juvenile offspring. Here, we investigated associations between WD-induced maternal metabolic and microbiome changes, in the absence of obesity, and miRNA and gene expression changes in the placenta and fetal liver. After ~8-11 months of WD feeding, dams were similar in body weight but exhibited mild, systemic inflammation (elevated CRP and neutrophil count) and dyslipidemia (increased triglycerides and cholesterol) compared with dams fed a control diet. The maternal gut microbiome was mainly comprised of Lactobacillales and Clostridiales, with significantly decreased alpha diversity (P = 0.0163) in WD-fed dams but no community-wide differences (P = 0.26). At 0.9 gestation, mRNA expression of IL6 and TNF in maternal WD (mWD) exposed placentas trended higher, while increased triglycerides, expression of pro-inflammatory CCR2, and histological evidence for fibrosis were found in mWD-exposed fetal livers. In the mWD-exposed fetus, hepatic expression levels of miR-204-5p and miR-145-3p were significantly downregulated, whereas in mWD-exposed placentas, miR-182-5p and miR-183-5p were significantly decreased. Notably, miR-1285-3p expression in the liver and miR-183-5p in the placenta were significantly associated with inflammation and lipid synthesis pathway genes, respectively. Blautia and Ruminococcus were significantly associated with miR-122-5p in liver, while Coriobacteriaceae and Prevotellaceae were strongly associated with miR-1285-3p in the placenta; both miRNAs are implicated in pathways mediating postnatal growth and obesity. Our findings demonstrate that mWD shifts the maternal microbiome, lipid metabolism, and inflammation prior to obesity and are associated with epigenetic changes in the placenta and fetal liver. These changes may underlie inflammation, oxidative stress, and fibrosis patterns that drive NAFLD and metabolic disease risk in the next generation.