A therapeutic vaccine strategy to prevent Pneumocystis pneumonia in an immunocompromised host in a non-human primate model of HIV and Pneumocystis co-infection.

Introduction: Pneumocystis is a ubiquitous fungal pathogen that causes pneumonia (PCP) and pulmonary sequelae in HIV-infected individuals and other immunocompromised populations. With the success of anti-retroviral therapy for HIV-infected individuals the frequency of PCP in that population has decreased, however, PCP remains a significant cause of morbidity and mortality in individuals with hematologic and solid malignancies, and in individuals treated with immunosuppressive therapies for autoimmune diseases, and following bone marrow and solid organ transplantation. Despite the clinical need, there is no approved vaccine to prevent PCP in vulnerable populations. The ultimate goal of the field is to develop an effective vaccine that can overcome immune deficits in at risk populations and induce long-lasting protective immunity to Pneumocystis. Toward this goal, our laboratory has established a model of PCP co-infection in simian immunodeficiency virus (SIV)-infected non-human primates (NHP) and identified a recombinant protein sub-unit vaccine, KEX1, that induces robust anti-Pneumocystis immunity in immune-competent macaques that is durable and prevents PCP following simian immunodeficiency virus (SIV)-induced immunosuppression. Type I, or invariant natural killer T (iNKT) cells have the potential to provide B cell help under conditions of reduced CD4+ T cell help. Methods: In the present study, we used the SIV model of HIV infection to address whether therapeutic vaccination with the iNKT cell-activating adjuvant α-galactosylceramide (α-GC) and KEX1 (α-GC+KEX1) can effectively boost anti-Pneumocystis humoral immunity following virus-induced immunosuppression. Results: Immunization of antigen-experienced NHPs with α-GC+KEX1 during the early chronic phase of SIV-infection significantly boosted anti-Pneumocystis humoral immunity by increasing memory B cells and antibody titers, and enhanced titer durability during SIV-induced immunosuppression. This therapeutic vaccination strategy boosted anti-Pneumocystis immune responses during SIV-infection and contributed to protection against Pneumocystis co-infection in KEX1-vaccinated macaques. Conclusion: These studies present a novel strategy for stimulating durable anti-Pneumocystis humoral immunity in the context of complex, chronic SIV-induced immunosuppression and may be further applied to immunization of other immunosuppressed populations, and toward other common recall antigens.

Prevalence and Healthcare Burden of Fungal Infections in the United States, 2018.

BACKGROUND: Fungal infections are responsible for >1.5 million deaths globally per year, primarily in those with compromised immune function. This is concerning as the number of immunocompromised patients, especially in those without human immunodeficiency virus (HIV), has risen in the past decade. The purpose of this analysis was to provide the current prevalence and impact of fungal disease in the United States. METHODS: We analyzed hospital discharge data from the most recent (2018) Healthcare Cost and Utilization Project National Inpatient Sample, and outpatient visit data from the National Ambulatory Medical Care Survey and the National Hospital Ambulatory Medical Care Survey. Costs are presented in 2018 United States (US) dollars. RESULTS: In the 35.5 million inpatient visits documented in 2018 in the US, approximately 666 235 fungal infections were diagnosed, with an estimated attributable cost of $6.7 billion. Aspergillus, Pneumocystis, and Candida infections accounted for 76.3% of fungal infections diagnosed, and 81.1% of associated costs. Most fungal disease occurred in patients with elevated risk of infection. The visit costs, lengths of stay, and risks of mortality in this population were more than twice that of those without fungal diagnoses. A further 6.6 million fungal infections were diagnosed during outpatient visits. CONCLUSIONS: Fungal disease is a serious clinical concern with substantial healthcare costs and significant increases in morbidity and mortality, particularly among predisposed patients. Increased surveillance, standardized treatment guidelines, and improvement in diagnostics and therapeutics are needed to support the rising numbers of at-risk patients.

Mortality Trends in Risk Conditions and Invasive Mycotic Disease in the United States, 1999-2018.

BACKGROUND: Invasive fungal infections in the United States are chronically underdiagnosed and a lack of coordinated surveillance makes the true burden of disease difficult to determine. The purpose of this analysis was to capture mortality-associated burden of risk conditions and fungal infections. METHODS: We analyzed data from the National Vital Statistics System from 1999 through 2018 to estimate the mortality attributed to risk conditions and related fungal disease. RESULTS: The number of risk conditions associated with fungal disease is steadily rising in the United States, with 1 047 422 diagnoses at time of death in 2018. While fungal disease decreased substantially from 1999 to 2010, primarily due to the control of human immunodeficiency virus (HIV) infection, the number of deaths with fungal diagnosis has increased in the non-HIV cohort, with significant increases in patients with diabetes, cancer, immunosuppressive disorders, or sepsis. CONCLUSIONS: The landscape of individuals at risk for serious fungal diseases is changing, with a continued decline in HIV-associated incidence but increased diagnoses in patients with cancer, sepsis, immunosuppressive disorders, and influenza. Additionally, there is an overall increase in the number of fungal infections in recent years, indicating a failure to control fungal disease mortality in these new immunocompromised cohorts. Improvement in the prevention and management of fungal diseases is needed to control morbidity and mortality in the rising number of immunocompromised and at-risk patients in the United States.

Immunogenicity and protective efficacy of a pan-fungal vaccine in preclinical models of aspergillosis, candidiasis, and pneumocystosis.

Invasive fungal infections cause over 1.5 million deaths worldwide. Despite increases in fungal infections as well as the numbers of individuals at risk, there are no clinically approved fungal vaccines. We produced a "pan-fungal" peptide, NXT-2, based on a previously identified vaccine candidate and homologous sequences from Pneumocystis, Aspergillus,Candida, and Cryptococcus. We evaluated the immunogenicity and protective capacity of NXT-2 in murine and nonhuman primate models of invasive aspergillosis, systemic candidiasis, and pneumocystosis. NXT-2 was highly immunogenic and immunized animals had decreased mortality and morbidity compared to nonvaccinated animals following induction of immunosuppression and challenge with Aspergillus, Candida, or Pneumocystis. Data in multiple animal models support the concept that immunization with a pan-fungal vaccine prior to immunosuppression induces broad, cross-protective antifungal immunity in at-risk individuals.

Vaccine-Induced Protection in Two Murine Models of Invasive Pulmonary Aspergillosis.

Life-threatening, invasive fungal infections (IFIs) cause over 1.5 million deaths worldwide and are a major public health concern with high mortality rates even with medical treatment. Infections with the opportunistic fungal pathogen, Aspergillus fumigatus are among the most common. Despite the growing clinical need, there are no licensed vaccines for IFIs. Here we evaluated the immunogenicity and protective efficacy of an A. fumigatus recombinant protein vaccine candidate, AF.KEX1, in experimental murine models of drug-induced immunosuppression. Immunization of healthy mice with AF.KEX1 and adjuvant induced a robust immune response. Following AF.KEX1 or sham immunization, mice were immunosuppressed by treatment with either cortisone acetate or hydrocortisone and the calcineurin inhibitor, tacrolimus. To test vaccine efficacy, immunosuppressed mice were intranasally challenged with A. fumigatus conidia (Af293) and weight and body temperature were monitored for 10 days. At study termination, organism burden in the lungs was evaluated by quantitative PCR and Gomori's methanamine silver staining. In both models of immunosuppression, AF.KEX1 vaccinated mice experienced decreased rates of mortality and significantly lower lung organism burden compared to non-vaccinated controls. The lung fungal burden was inversely correlated with the peak anti-AF.KEX1 IgG titer achieved following vaccination. These studies provide the basis for further evaluation of a novel vaccine strategy to protect individuals at risk of invasive aspergillosis due to immunosuppressive treatments.

Relationship of Pneumocystis antibody responses to paediatric asthma severity.

BACKGROUND: Although asthma is the most commonly diagnosed respiratory disease, its pathogenesis is complex, involving both genetic and environmental factors. A role for the respiratory microbiome in modifying asthma severity has been recently recognised. Airway colonisation by Pneumocystis jirovecii has previously been associated with multiple chronic lung diseases, including chronic obstructive pulmonary disease (COPD) and severe asthma (SA). Decreased incidence of Pneumocystis pneumonia in HIV-infected individuals and reduced severity of COPD is associated with naturally occurring antibody responses to the Pneumocystis antigen, Kexin (KEX1). METHODS: 104 paediatric patients were screened for KEX1 IgG reciprocal end point titre (RET), including 51 with SA, 20 with mild/moderate asthma, 20 non-asthma and 13 with cystic fibrosis (CF) in a cross-sectional study. RESULTS: Patients with SA had significantly reduced Pneumocystis KEX1 titres compared with patients with mild/moderate asthma (p=0.018) and CF (p=0.003). A binary KEX1 RET indicator was determined at a threshold of KEX1 RET=1000. Patients with SA had 4.40 (95% CI 1.28 to 13.25, p=0.014) and 17.92 (95% CI 4.15 to 66.62, p<0.001) times the odds of falling below that threshold compared with mild/moderate asthma and patients with CF, respectively. Moreover, KEX1 IgG RET did not correlate with tetanus toxoid IgG (r=0.21, p=0.82) or total IgE (r=0.03, p=0.76), indicating findings are specific to antibody responses to KEX1. CONCLUSIONS: Paediatric patients with SA may be at higher risk for chronic Pneumocystis infections and asthma symptom exacerbation due to reduced levels of protective antibodies. Plasma KEX1 IgG titre may be a useful parameter in determining the clinical course of treatment for paediatric patients with asthma.

Tobacco Use Among Rural and Urban US Middle and High School Students: National Youth Tobacco Survey, 2011-2016.

PURPOSE: Rural adolescents engage in higher smoking and smokeless tobacco use rates than those from urban communities; urban adolescents are more likely to use e-cigarettes. The study investigated whether place of residence (rural vs urban) is associated with tobacco use prevalence and change in prevalence among middle and high school students over time. METHODS: We analyzed data from the National Youth Tobacco Survey (2011-2016). Multiple logistic regression methods for weighted survey data assessed the relationship of place of residence with current tobacco product use over time, adjusting for demographics. FINDINGS: There was no difference in rate of change in use of any tobacco product between rural and urban middle or high school students. Adjusting for age, sex, race/ethnicity, and survey year, both middle and high school rural students were more likely to use cigarettes and smokeless tobacco, whereas urban high school students were more likely to use hookah. Significant polynomial trends were observed for e-cigarette and hookah use patterns, whereas linear changes in use patterns were detected for cigarette and smokeless tobacco use over time. CONCLUSIONS: Rural high school students are more likely to smoke cigarettes and use smokeless tobacco than their urban counterparts, although prevalence rates have decreased over time. However, use of hookah and e-cigarettes among middle and high school students has increased over time regardless of place of residence. To stem the rapid increase in use of hookah and e-cigarettes, comprehensive tobacco control policies are needed regardless of rural or urban location.

Statin treatment prevents the development of pulmonary arterial hypertension in a nonhuman primate model of HIV-associated PAH.

Pulmonary arterial hypertension (PAH) is a life-threatening disease characterized by pulmonary vascular remodeling, elevated pulmonary arterial pressure, and right heart failure. Human immunodeficiency virus (HIV)-infected individuals have a higher incidence of PAH than the non-HIV infected population and evidence suggests a role for systemic and pulmonary inflammation in the pathogenesis of HIV-associated PAH. Due to their pleiotropic effects, including immune-modulatory and anti-inflammatory effects, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) have been considered for the treatment of PAH, with conflicting results. The effects of statins on HIV-associated PAH have not been specifically evaluated. We have developed a non-human primate (NHP) model of HIV-associated PAH that closely mimics HIV-PAH using simian immunodeficiency virus (SIV)-infected rhesus macaques (Macaca mulatta). We determined that treatment of healthy macaques with atorvastatin prior to and throughout SIV infection prevented the development of SIV-associated PAH. Additionally, SIV-infected macaques that initiated atorvastatin treatment during the early chronic disease stage had reduced incidence of PAH compared to untreated animals. Statin treatment reduced inflammatory mediators TGF-ß, MIP-1α, and TNF-α and the numbers of CD14dimCD16+ non-classical monocytes, and CD14+CCR7-CD163-CD206+ alveolar macrophages previously shown to be associated with SIV-PAH. These results support the concept that statins reduce inflammatory processes that contribute to PAH and may provide a safe and effective prophylactic strategy for the prevention of PAH in HIV-infected individuals.

Immunization with Pneumocystis recombinant KEX1 induces robust and durable humoral responses in immunocompromised non-human primates.

Infection with the opportunistic fungal pathogen, Pneumocystis jirovecii causes life-threatening pneumonia in immunocompromised individuals. In addition to HIV-1 infected patients, individuals at risk of Pneumocystis infection include those receiving immunosuppressive therapies due to transplantation, cancer or autoimmune disease. Antibiotic treatment is not always successful, and it does not prevent obstructive lung disease after clearance of the pathogen. Therefore, it is essential to develop therapeutic alternatives that are more effective against PCP. We reported that Pneumocystis recombinant protein KEX1 induces protective immunity against the development of PCP in a non-human primate model of HIV-induced immunosuppression. In this study, we tested the immunogenicity KEX1 immunization of healthy rhesus macaques and the durability of these responses during drug-induced immunosuppression using tacrolimus (FK506) and methylprednisolone. We observed that vaccination with KEX1 prior to the start of the immunosuppressive regimen generated a robust and long-lasting antibody response that was maintained throughout the immunosuppressive treatment. Furthermore, boosting with KEX1 during immunosuppression induced recall of memory responses against recombinant KEX1. The durability of the anti-KEX1 response and the ability to induce a recall response during immunosuppressive therapy provide a proof-of-concept data supporting further investigation of the KEX1 as a prophylactic vaccine to prevent PCP in drug-induced immunosuppression. This approach provides fundamental knowledge for the elaboration of therapeutic and prophylactic alternatives for PCP in patients undergoing severe immunosuppressive therapy.

The Gene CG6767 Affects Olfactory Behavior in Drosophila melanogaster.

Chemosensory systems mediate some of the most vital animal behaviors. However, our knowledge of the genetic mechanisms that underlie behavioral responses to olfactory cues remains fragmented. Genome-wide association mapping has greatly advanced our ability to identify candidate loci associated with variation in olfactory behavior, but functional validation of these candidates remain a necessary next step in understanding the mechanisms by which these genes influence chemoreception. In previous genome-wide association analyses, a genomic region that spans multiple polymorphic loci on the left arm of the third chromosome was found to be significantly associated with variation in olfactory behavioral responses to the odorant 2,3-butanedione, a volatile compound present in fermenting fruit. In this study, behavioral analysis of flies possessing either the major or minor haplotype for this region confirmed the association between polymorphisms in the region and variation in olfactory behavior. Moreover, functional dissection of the genes within this region using P-element insertional mutagenesis together with targeted RNAi experiments revealed that the gene CG6767, a gene of previously unknown function but predicted to encode an enzyme responsible for the synthesis and metabolism of nucleic acids, affects olfactory behavioral responses to 2,3-butanedione. Specifically, RNAi mediated knockdown of CG6767 expression in different neuroanatomical populations of the olfactory system suggests that this gene functions in local interneurons of the antennal lobe. These results reveal a new role for CG6767 and its importance in olfactory behavior.

Monocyte and Alveolar Macrophage Skewing Is Associated with the Development of Pulmonary Arterial Hypertension in a Primate Model of HIV Infection.

We investigated the relationship of monocytes, alveolar, and tissue-resident macrophage populations and the development of pulmonary arterial hypertension (PAH) in a nonhuman primate model of HIV infection. A prospective study of simian immunodeficiency virus-associated pulmonary arterial hypertension (SIV-PAH) was done. Rhesus macaques (n = 21) were infected with SIV. Blood, bronchoalveolar lavage fluid (BALF), and lung tissue were analyzed for monocyte and macrophage phenotypes and inflammatory mediators. Serial right heart catheterizations were performed at three time points throughout the study to assess hemodynamic alterations and the development of PAH. All 21 animals showed similar courses of SIV infection with an increasing proinflammatory plasma environment. At 6 months postinfection (mpi), 11 of 21 animals developed SIV-PAH (mPAP ≤25 mmHg; right ventricular systolic pressure [RVSP] ≤36 mmHg). PAH+ animals had an increased frequency of proinflammatory, nonclassical monocytes (CD14dimCD16+) (p = .06) in the peripheral blood and CD14+CCR7-CD163-CD206+ macrophages (p = .04) in BALF compared with PAH- animals at 6 mpi. Increased frequencies of these monocyte and macrophage phenotypes correlated with elevated RVSP (p = .04; p = .03). In addition, PAH+ animals had greater frequencies of tissue resident inflammatory M1-like CD68+STAT1+ (p = .001) and M2a-like CD68+STAT3+ macrophages (p = .003) and a lower frequency of anti-inflammatory M2c-like CD68+STAT6+ macrophages (p = .003) as well as fewer interleukin (IL)-10+ cells (p = .01). The results suggest that HIV-PAH is associated with skewing of monocytes and alveolar macrophages toward a proinflammatory, profibrotic phenotype. Furthermore, PAH+ animals may have diminished capacity to downregulate exaggerated chronic inflammation, as indicated by lower levels of IL-10 in PAH+ animals, contributing to disease progression.

Longitudinal Evaluation of Pulmonary Arterial Hypertension in a Rhesus Macaque (Macaca mulatta) Model of HIV Infection.

Pulmonary arterial hypertension (PAH) is a life-threatening disease with higher incidence in HIV-infected compared with noninfected patients. SIV-infected NHP develop clinical manifestations of HIV infection, including PAH. To understand the pathogenesis of PAH and determine the relationship between hemodynamic changes and clinical characteristics associated with SIV infection, we performed right heart catheterization and echocardiographic imaging of 21 rhesus macaques before and after SIV infection. Between 6 and 12 mo after infection, 11 of the 21 animals had elevated mean pulmonary arterial pressure (mPAP; greater than 25 mm Hg). RV involvement was evident as increased RV glucose uptake in PAH+ macaques on positron emission tomography-coupled CT compared with uninfected animals. RV and pulmonary vascular collagen deposition were elevated in PAH+ animals. At 12 mo after infection, 6 of the 21 macaques (28.6%) exhibited continued increase in mPAP (progressive PAH), whereas 5 animals (23.8%) had reduced pressure (transient PAH). SIV infection of rhesus macaques led to 3 distinct outcomes with regard to hemodynamic function. Hemodynamic alterations correlated with specific inflammatory profiles and increased RV and pulmonary arterial fibrosis but not with viral load, sex, or CD4+ T-cell levels. This model of a natural cause of PAH provides insight into disease pathways that are important for the development of novel therapeutic targets.

Associations of Demographic Factors and Tobacco Use With Progesterone and Estradiol During Pregnancy.

OBJECTIVE: To evaluate the association of biochemically validated prenatal tobacco use with serum progesterone and estradiol in the second trimester of pregnancy, controlling for demographic and personal factors. STUDY DESIGN: This secondary analysis of a multicenter longitudinal study included 114 women with singleton pregnancies. Multiple regression analysis assessed whether prenatal tobacco use was related to hormone levels during the second trimester, controlling for covariates (age, body mass index, and race or ethnicity, with gestational age added to subsequent models). RESULT: In the initial regressions, tobacco users had significantly lower progesterone level compared with nonsmokers (p = .037), while estradiol was unrelated to prenatal tobacco use. Women with greater body mass index also had significantly lower progesterone (p = .028), but body mass index was unrelated to estradiol. With gestational age as an additional covariate, prenatal tobacco use was no longer a significant predictor of progesterone, but both body mass index and gestational age were significant (F = 10.6, p < .001, R 2 = 0.35). For estradiol, the overall regression of estradiol on age, body mass index, and race or ethnicity was not significant (F = 1.2, p = .31). With gestational age added to the model, the overall model was significant (F = 7.2, p < .001, R 2 = 0.27). CONCLUSION: This study provides additional evidence that prenatal tobacco use may influence lower serum progesterone during the second trimester. This is of particular concern given the link between depressed progesterone activity and risk for preterm birth.

Current Use of E-Cigarettes and Conventional Cigarettes Among US High School Students in Urban and Rural Locations: 2014 National Youth Tobacco Survey.

PURPOSE: Adolescent tobacco use is higher in rural than in urban areas. While e-cigarette use is increasing rapidly among this age group, differences in prevalence between rural versus urban populations for this relatively novel product have not been explored. The purpose is to investigate whether location of school (rural-urban) is associated with e-cigarette use and dual use (defined as the use of both e-cigarettes and conventional cigarettes) among high school students. DESIGN: Cross-sectional survey obtained using a stratified, 3-stage cluster sample design. SETTING: United States. PARTICIPANTS: A nationally representative sample of US high school students (N = 11 053) who completed the 2014 National Youth Tobacco Survey (NYTS); slightly more than half were urban (54%). MEASURES: The NYTS measures tobacco-related knowledge, attitudes, and use behavior and demographics of students in the United States. ANALYSIS: Weighted logistic regression assessed the relationships of urban-rural location with current e-cigarette use and dual use, adjusting for demographic factors, perceived risk, and social norms. RESULTS: There were clear differences in patterns of adolescent e-cigarette and cigarette use in rural versus urban areas. Social norms and perceptions may play a role in understanding these differences. CONCLUSION: Urban youth current cigarette smokers were nearly twice as likely as rural cigarette smokers to also use e-cigarettes. Reasons for urban-rural differences need to be taken into account when designing prevention programs and policy changes.

Advertising exposure and use of e-cigarettes among female current and former tobacco users of childbearing age.

OBJECTIVE: The study examined the relationship between exposure to e-cigarette advertising and e-cigarette use by pregnancy status, including use of flavored e-cigarette products, among women of childbearing age. DESIGN: A cross-sectional, correlational design was used. SUBJECTS: Female current or former tobacco users in Central and Eastern Kentucky, 18-45 years old (N = 194, 52% pregnant). MEASURES: Demographics, pregnancy status, cigarette and e-cigarette use, and exposure to e-cigarette advertising. RESULTS: Younger age, white non-Hispanic race, and greater exposure to e-cigarette advertising were associated with a higher likelihood of ever using e-cigarettes (p < .05 for each variable). Pregnancy was not associated with ever use (p = .11). Younger age was associated with use of flavored e-cigarettes (p = .0027). Among e-cigarette users, those who used flavored products were more likely to have seen advertisements or information about e-cigarettes on social media, compared to those who used unflavored e-cigarettes only (p = .016). CONCLUSION: There is a link between advertising exposure and ever use of e-cigarettes. Pregnancy status is not significantly associated with ever use. Use of flavored e-cigarettes is associated with younger age. E-cigarette users with greater exposure to advertising on social media were more likely to use flavored products.

Perinatal Biochemical Confirmation of Smoking Status by Trimester.

INTRODUCTION: Tobacco use during pregnancy is the most modifiable risk factor associated with poor pregnancy outcomes. Self-reported tobacco use has been demonstrated to have high misclassification rates. The aims were to examine misclassification rates of perinatal tobacco use during each trimester of pregnancy and 8 weeks postpartum, and to evaluate characteristics associated with misclassification of tobacco use status. METHODS: This is secondary analysis of a prospective, multicenter trial of pregnant women, and it includes participants who were biochemically identified as tobacco users during their first trimester (N = 103). Each trimester and once postpartum, tobacco use was assessed via self-report and validated using a cutoff of 100 ng/mL for urine cotinine via NicAlert test strips to indicate current use. Those who self-reported as nonusers but were identified as users via urine cotinine were considered misclassified; misclassification rates were determined for each time period. Logistic regression assessed maternal factors associated with misclassification status. RESULTS: Misclassification rates declined from 35.0% at first trimester to 31.9% and 26.6% at the second and third; the postpartum rate was 30.4%. These rates did not differ significantly from each other at the 0.05 level. Race/ethnicity was associated with misclassification status; white/non-Hispanic women were 87% less likely to be misclassified (p < .001). CONCLUSION: Misclassification of prenatal smoking status decreases as pregnancy progresses, though the observed rate change was not significant. Minority women may be at particular risk for non-disclosure of tobacco use. Biochemical validation should be considered when assessing perinatal tobacco use via self-report, given high misclassification rates throughout the perinatal period. IMPLICATIONS: These results demonstrate that regardless of trimester, more than one-quarter of tobacco-using pregnant women may not disclose tobacco use throughout pregnancy and early postpartum. Although the rate of misclassification decreased from first to third trimester and then increased in the immediate postpartum, these changes in misclassification rates were not significant. Minority groups may be at particular risk of misclassification compared with white/non-Hispanic women. Biochemical validation is warranted throughout pregnancy to encourage cessation as tobacco use is one of the most easily-modified risk factors for poor birth outcomes.