Design of a randomized, placebo-controlled study evaluating efficacy and safety of a cancer preventative vaccine in dogs.

Preventative anti-cancer vaccination strategies have long been hampered by the challenge of targeting the diverse array of potential tumor antigens, with successes to date limited to cancers with viral etiologies. Identification and vaccination against frameshift neoantigens conserved across multiple species and tumor histologies is a potential cancer preventative strategy currently being investigated. Companion dogs spontaneously develop cancers at a similar incidence to those in people and are a complementary comparative patient population for the development of novel anti-cancer therapeutics. In addition to an intact immune system with tumors that arise in an autochthonous tumor microenvironment, dogs also have a shorter lifespan and temporally compressed tumor natural history as compared to humans, which allows for more rapid evaluation of safety, immunogenicity, and efficacy of cancer vaccination strategies. Here we describe the study protocol for the Vaccination Against Canine Cancer Study (VACCS), the largest interventional cancer clinical trial conducted in companion dogs to date. In addition to safety and immunogenicity, the primary endpoint of VACCS is the cumulative incidence (CI) of dogs developing malignant neoplasia of any type at the end of the study period. Secondary endpoints include changes in incidence of specific tumor types, survival times following neoplasia diagnosis, and all-cause mortality.

Observational study of effects of HIV Acquisition and Antiretroviral Treatment on Biomarkers of Systemic Immune Activation.

Objective: Assess whether biomarkers of systemic inflammation are associated with HIV acquisition or with the timing of ART initiation ("immediate", at diagnosis, versus "deferred", at 24 weeks post-diagnosis) in men-who-have-sex-with-men (MSM) and transgender women. Design: A retrospective study comparing inflammatory biomarkers in participants' specimens collected before and after ≥2 years of effective ART. Methods: Inflammatory biomarkers were measured in four longitudinally collected plasma specimens, including two plasma specimens collected from each participant before and two after HIV acquisition and confirmed ART-suppression. Biomarkers were quantified by enzyme-linked immuno-assay or Meso Scale Discovery. Statistical measures compared intra-participant and between-group changes in biomarkers. Results: Across 50 participants, the levels of C-reactive protein (CRP), monocyte chemo-attractant protein-1, tumor necrosis factor-α and interferon gamma-induced protein-10 significantly increased while leptin and lipopolysaccharide binding protein (LBP) significantly decreased following HIV infection. Randomization to deferred-ART initiation was associated with greater increases in CRP and no decreases in LBP. Multiple biomarkers varied significantly within participants' two pre-infection or two post-ART-suppression specimens. Conclusions: Acquisition of HIV appeared to induce systemic inflammation, with elevation of biomarkers previously associated with infections and cardiovascular disease. Initiation of ART during the early weeks of infection tempered the increase in pro-inflammatory biomarkers compared to those who delayed ART for ~24 weeks after HIV diagnosis, perhaps because immediate-ART limited the size of the HIV reservoir or limited immune dysregulation. Some but not all biomarkers appeared sufficiently stable to assess intraparticipant changes over time. Given that pro-inflammatory biomarkers predict multiple co-morbidities, our findings suggest that immediate-ART initiation may improve health outcomes.

Low rate of SARS-CoV-2 incident infection identified by weekly screening PCR in a prospective year-long cohort study.

BACKGROUND: Asymptomatic and pre-symptomatic SARS-CoV-2 infections may contribute to ongoing community transmission, however, the benefit of routine screening of asymptomatic individuals in low-risk populations is unclear. METHODS: To identify SARS-CoV-2 infections 553 seronegative individuals were prospectively followed for 52 weeks. From 4/2020-7/2021, participants submitted weekly self-collected nasal swabs for rtPCR and completed symptom and exposure surveys. RESULTS: Incident SARS2-CoV-2 infections were identified in 9/553 (1.6%) participants. Comparisons of SARS2-CoV-2(+) to SARS2-CoV-2(-) participants revealed significantly more close contacts outside the household (median: 5 versus 3; p = 0.005). The incidence of infection was higher among unvaccinated/partially vaccinated than among fully vaccinated participants (9/7,679 versus 0/6,845 person-weeks; p = 0.004). At notification of positive test result, eight cases were symptomatic and one pre-symptomatic. CONCLUSIONS: These data suggest that weekly SARS2-CoV2 surveillance by rtPCR did not efficiently detect pre-symptomatic infections in unvaccinated participants.

Angiotensin II receptor I auto-antibodies following SARS-CoV-2 infection.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is associated with endothelial activation and coagulopathy, which may be related to pre-existing or infection-induced pro-thrombotic autoantibodies such as those targeting angiotensin II type I receptor (AT1R-Ab). METHODS: We compared prevalence and levels of AT1R-Ab in COVID-19 cases with mild or severe disease to age and sex matched negative controls utilizing multivariate logistic and quantile regression adjusted for comorbidities including hypertension, diabetes, and heart disease. RESULTS: There were trends toward increased prevalence (50% vs. 33%, p = 0.1) and level of AT1R-Ab (median 9.8 vs. 6.1 U/mL, p = 0.06) in all cases versus controls. When considered by COVID-19 disease severity, there was a trend toward increased prevalence of AT1R-Ab (55% vs. 31%, p = 0.07), as well as significantly higher AT1R-Ab levels (median 10.7 vs. 5.9 U/mL, p = 0.03) amongst individuals with mild COVID-19 versus matched controls. In contrast, the prevalence (42% vs. 37%, p = 0.9) and level (both medians 6.7 U/mL, p = 0.9) of AT1R-Ab amongst those with severe COVID-19 did not differ from matched controls. CONCLUSIONS: These findings support an association between COVID-19 and AT1R-Ab, emphasizing that vascular pathology may be present in individuals with mild COVID-19 as well as those with severe disease.

Central obesity is a contributor to systemic inflammation and monocyte activation in virally suppressed adults with chronic HIV in Kenya.

OBJECTIVES: Heightened systemic inflammation is common in obese individuals and persons with HIV (PWH) and is independently associated with an increased risk of cardiovascular diseases (CVDs). We investigated the combined effect of central obesity, a surrogate measure of visceral fat and HIV on circulating levels of inflammatory cytokines among Kenyan adults. DESIGN: A cross-sectional study. METHODS: We analysed and compared data from 287 virally suppressed PWH and 277 noninfected Kenyan adults, including biomarkers of gut epithelial dysfunction (intestinal fatty acid binding protein), monocyte activation (soluble CD163 and CD14) and inflammation [interleukin (IL)-1ß, IL-6, TNF-α and hsCRP] by HIV/central obesity status (HIV-positive/obese, HIV-negative/obese, HIV-positive/nonobese and HIV-negative/nonobese). Central obesity was defined as waist circumference more than 80 cm for women and more than 94 cm for men. We assessed the association of HIV/obesity status with elevated biomarkers (>75th percentile) using logistic regression. RESULTS: Median age for participants was 44 years and 37% were centrally obese. Levels of all biomarkers were higher among the HIV-positive/obese compared with the HIV-negative/nonobese (P < 0.05 for all comparisons). The HIV-positive/obese group had the greatest odds of having elevated inflammatory biomarkers compared with other groups even after adjustment of age, BMI and other conventional CVD risk factors (P < 0.05 for all). Additional adjustment for sCD163 in the multivariate model substantially attenuated the association for HIV-positive/obesity with IL-1ß, IL-6 and TNF-α but not hsCRP. The contribution of HIV-positive/obesity to inflammation was independent of the degree of immunosuppression. CONCLUSION: Central obesity is prevalent among virally suppressed African PWH and is associated with greater inflammation and monocyte activation independent of other comorbidities and HIV-specific factors.

Rapid decline of neutralizing antibodies is associated with decay of IgM in adults recovered from mild COVID-19.

The fate of protective immunity following mild severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection remains ill defined. Here, we characterize antibody responses in a cohort of participants recovered from mild SARS-CoV-2 infection with follow-up to 6 months. We measure immunoglobulin A (IgA), IgM, and IgG binding and avidity to viral antigens and assess neutralizing antibody responses over time. Furthermore, we correlate the effect of fever, gender, age, and time since symptom onset with antibody responses. We observe that total anti-S trimer, anti-receptor-binding domain (RBD), and anti-nucleocapsid protein (NP) IgG are relatively stable over 6 months of follow-up, that anti-S and anti-RBD avidity increases over time, and that fever is associated with higher levels of antibodies. However, neutralizing antibody responses rapidly decay and are strongly associated with declines in IgM levels. Thus, while total antibody against SARS-CoV-2 may persist, functional antibody, particularly IgM, is rapidly lost. These observations have implications for the duration of protective immunity following mild SARS-CoV-2 infection.

Myeloid-derived suppressor cells and their association with vaccine immunogenicity in South African infants.

The role of Myeloid-Derived Suppressor Cells (MDSC) in infant immune ontogeny is unknown. Here, we evaluated MDSC frequency and relationship with infant vaccine responses throughout the first year of life in a prospective cohort study. Ninety-one South African infant-mother pairs were enrolled at delivery, and blood samples were collected at 0, 6, 10, and 14 weeks, 6 months, 9 months, and 1 year. MDSC frequencies were quantified, and immune responses to the childhood vaccines Bacillus Calmette-Guérin (BCG), hepatitis B (HepB), and combination diphtheria, tetanus, and pertussis (dTaP) were measured by Ag-specific CD4+ T cell proliferation and interferon gamma (IFN-γ) production. Vaccine-specific Ab responses to HepB, dTaP, and Haemophilus influenzae type b (Hib) were quantified via Enzyme-Linked Immunosorbent assay (ELISA). MDSC frequency in mother-infant pairs was strongly correlated; the frequency of MDSC decreased in both mothers and infants during the months after delivery/birth; and by 1 year, infant MDSC frequencies rebounded to birth levels. Higher MDSC frequency at vaccination was associated with a lack of subsequent IFN-γ release in response to vaccine Ags, with the exception of BCG. With the exception of a weak, positive correlation between MDSC frequency at 6 weeks (time of initial vaccination) and peak Hepatitis B surface antigen Ab titer, Polymorphonuclear Myeloid-Derived Suppressor Cells (PMN-MDSC) was not correlated with T cell proliferation or Ab responses in this study. The potential for MDSC-mediated suppression of vaccine Ag-specific IFN-γ responses should be explored further, and considered when evaluating candidate infant vaccines.

Endothelial Dysfunction Is Related to Monocyte Activation in Antiretroviral-Treated People With HIV and HIV-Negative Adults in Kenya.

BACKGROUND: Residual monocyte activation may contribute to increased risk for endothelial dysfunction and subsequent atherosclerotic cardiovascular diseases (CVDs) among people with HIV (PWH) on antiretroviral therapy (ART). We examined the relationship between monocyte activation and endothelial activation in PWH in Kenya. METHODS: Serum levels of markers of endothelial activation (soluble/circulating intercellular [sICAM-1] and vascular [sVCAM-1] cell adhesion molecule-1), intestinal barrier dysfunction (intestinal fatty acid binding protein [I-FABP]), and monocyte activation (soluble CD14 [sCD14]) were measured in 275 PWH on ART and 266 HIV-negative persons. Linear regression was used to evaluate associations, adjusting for demographic and traditional CVD risk factors. RESULTS: Among 541 participants, the median age was 43 years, 50% were female, and most PWH were virally suppressed (97%). sICAM-1 and sVCAM-1 levels were significantly higher in PWH than in HIV-negative participants (P < .001 for both). After further adjustment for traditional CVD risk factors, HIV infection remained associated with 49% (95% CI, 33% to 67%) greater sICAM-1 and 30% (95% CI, 14% to 48%) greater sVCAM-1 relative to uninfected controls. Adjustment for sCD14 substantially attenuated the difference between PWH and HIV-negative individuals. In a stratified analysis of PWH, both sICAM-1 and sVCAM-1 were positively associated with sCD14 (P < .001). CONCLUSIONS: Despite viral suppression, African PWH have evidence of enhanced endothelial activation associated with sCD14, suggesting that monocyte activation plays a role in atherosclerotic plaque development. Future studies are needed to determine mechanistic pathways leading to monocyte activation in this population.

Kinetics of Myeloid-Derived Suppressor Cell Frequency and Function during Simian Immunodeficiency Virus Infection, Combination Antiretroviral Therapy, and Treatment Interruption.

During chronic lentiviral infection, poor clinical outcomes correlate both with systemic inflammation and poor proliferative ability of HIV-specific T cells; however, the connection between the two is not clear. Myeloid-derived suppressor cells (MDSC), which expand during states of elevated circulating inflammatory cytokines, may link the systemic inflammation and poor T cell function characteristic of lentiviral infections. Although MDSC are partially characterized in HIV and SIV infection, questions remain regarding their persistence, activity, and clinical significance. We monitored MDSC frequency and function in SIV-infected rhesus macaques. Low MDSC frequency was observed prior to SIV infection. Post-SIV infection, MDSC were elevated in acute infection and persisted during 7 mo of combination antiretroviral drug therapy (cART). After cART interruption, we observed MDSC expansion of surprising magnitude, the majority being granulocytic MDSC. At all stages of infection, granulocytic MDSC suppressed CD4+ and CD8+ T cell proliferation in response to polyclonal or SIV-specific stimulation. In addition, MDSC frequency correlated significantly with circulating inflammatory cytokines. Acute and post-cART levels of viremia were similar, however, the levels of inflammatory cytokines and MDSC were more pronounced post-cART. Expanded MDSC during SIV infection, especially during the post-cART inflammatory cytokine surge, likely limit cellular responses to infection. As many HIV curative strategies require cART interruption to determine efficacy, our work suggests treatment interruption-induced MDSC may especially undermine the effectiveness of such strategies. MDSC depletion may enhance T cell responses to lentiviral infection and the effectiveness of curative approaches.

Is Infant Immunity Actively Suppressed or Immature?

Almost 7 million children under the age 5 die each year, and most of these deaths are attributable to vaccine-preventable infections. Young infants respond poorly to infections and vaccines. In particular, dendritic cells secrete less IL-12 and IL-18, CD8pos T cells and NK cells have defective cytolysis and cytokine production, and CD4pos T cell responses tend to bias towards a Th2 phenotype and promotion of regulatory T cells (Tregs). The basis for these differences is not well understood and may be in part explained by epigenetic differences, as well as immaturity of the infant's immune system. Here we present a third possibility, which involves active suppression by immune regulatory cells and place in context the immune suppressive pathways of mesenchymal stromal cells (MSC), myeloid-derived suppressor cells (MDSC), CD5pos B cells, and Tregs. The immune pathways that these immune regulatory cells inhibit are similar to those that are defective in the infant. Therefore, the immune deficiencies seen in infants could be explained, in part, by active suppressive cells, indicating potential new avenues for intervention.

Myeloid derived suppressor cells are present at high frequency in neonates and suppress in vitro T cell responses.

Over 4 million infants die each year from infections, many of which are vaccine-preventable. Young infants respond relatively poorly to many infections and vaccines, but the basis of reduced immunity in infants is ill defined. We sought to investigate whether myeloid-derived suppressor cells (MDSC) represent one potential impediment to protective immunity in early life, which may help inform strategies for effective vaccination prior to pathogen exposure. We enrolled healthy neonates and children in the first 2 years of life along with healthy adult controls to examine the frequency and function of MDSC, a cell population able to potently suppress T cell responses. We found that MDSC, which are rarely seen in healthy adults, are present in high numbers in neonates and their frequency rapidly decreases during the first months of life. We determined that these neonatal MDSC are of granulocytic origin (G-MDSC), and suppress both CD4+ and CD8+ T cell proliferative responses in a contact-dependent manner and gamma interferon production. Understanding the role G-MDSC play in infant immunity could improve vaccine responsiveness in newborns and reduce mortality due to early-life infections.

The role of myeloid-derived suppressor cells in immune ontogeny.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of granulocytic or monocytic cells that suppress innate as well as adaptive immune responses. In healthy adults, immature myeloid cells differentiate into macrophages, dendritic cells, and granulocytes in the bone marrow and MDSC are rarely detected in peripheral blood. However, in certain pathologies, in particular malignancies and chronic infection, differentiation of these cells is altered resulting in accumulation of circulating suppressive myeloid cells. MDSC express suppressive factors such as arginase-1, reactive oxygen species, and inducible nitric oxide synthase, which have the ability to inhibit T cell proliferation and cytoxicity, induce the expansion of regulatory T cells, and block natural killer cell activation. It is increasingly recognized that MDSC alter the immune response to several cancers, and perhaps chronic viral infections, in clinically important ways. In this review, we outline the potential contribution of MDSC to the generation of feto-maternal tolerance and to the ineffective immune responses to many infections and vaccines observed in early post-natal life. Granulocytic MDSC are present in large numbers in pregnant women and in cord blood, and wane rapidly during infancy. Furthermore, cord blood MDSC suppress in vitro T cell and NK responses, suggesting that they may play a significant role in human immune ontogeny. However, there are currently no data that demonstrate in vivo effects of MDSC on feto-maternal tolerance or immune ontogeny. Studies are ongoing to evaluate the functional importance of MDSC, including their effects on control of infection and response to vaccination in infancy. Importantly, several pharmacologic interventions have the potential to reverse MDSC function. Understanding the role of MDSC in infant ontogeny and their mechanisms of action could lead to interventions that reduce mortality due to early-life infections.

Global health: a successful context for precollege training and advocacy.

Despite a flourishing biomedical and global health industry too few of Washington state's precollege students are aware of this growing sector and emerging ideas on bacteria, fungi, parasites and viruses. Against the backdrop of numerous reports regarding declining precollege student interest in science, a precollege program was envisioned at Seattle Biomedical Research Institute (as of 2010, Seattle BioMed) to increase youth engagement in biomedical research and global health, increase community interest in infectious diseases and mobilize a future biomedical workforce. Since 2005, 169 rising high school juniors have participated in the BioQuest Academy precollege immersion program at Seattle BioMed. Assembling in groups of 12, students conduct laboratory experiments (e.g., anopheline mosquito dissection, gene expression informed tuberculosis drug design and optimizing HIV immunization strategies) related to global health alongside practicing scientific mentors, all within the footprint the institute. Laudable short-term impacts of the program include positive influences on student interest in global health (as seen in the students' subsequent school projects and their participation in Seattle BioMed community events), biomedical careers and graduate school (e.g., 16.9% of teens departing 2008-2009 Academy report revised goals of attaining a doctorate rather than a baccalaureate diploma). Long-term, 97% of alumni (2005-2008) are attending postsecondary schools throughout North America; eight graduates have already published scientific articles in peer-reviewed journals and/or presented their scientific data at national and international meetings, and 26 have been retained by Seattle BioMed researchers as compensated technicians and interns. Providing precollege students with structured access to practicing scientists and authentic research environments within the context of advancing global health has been a robust means of both building a future pool of talented leaders and engaged citizenry and increasing the visibility of health disparities within the community.

Differential regulation of inflammatory cytokine secretion by human dendritic cells upon Chlamydia trachomatis infection.

Chlamydia trachomatis is an obligate intracellular gram-negative bacterium responsible for a wide spectrum of diseases in humans. Both genital and ocular C. trachomatis infections are associated with tissue inflammation and pathology. Dendritic cells (DC) play an important role in both innate and adaptive immune responses to microbial pathogens and are a source of inflammatory cytokines. To determine the potential contribution of DC to the inflammatory process, human DC were infected with C. trachomatis serovar E or L2. Both C. trachomatis serovars were found to infect and replicate in DC. Upon infection, DC up-regulated the expression of costimulatory (B7-1) and cell adhesion (ICAM-1) molecules. Furthermore, chlamydial infection induced the secretion of interleukin-1beta (IL-1beta), IL-6, IL-8, IL-12p70, IL-18, and tumor necrosis factor alpha (TNF-alpha). The mechanisms involved in Chlamydia-induced IL-1beta and IL-18 secretion differed from those of the other cytokines. Chlamydia-induced IL-1beta and IL-18 secretion required infection with viable bacteria and was associated with the Chlamydia-induced activation of caspase-1 in infected host cells. In contrast, TNF-alpha and IL-6 secretion did not require that the Chlamydia be viable, suggesting that there are at least two mechanisms involved in the Chlamydia-induced cytokine secretion in DC. Interestingly, an antibody to Toll-like receptor 4 inhibited Chlamydia-induced IL-1beta, IL-6, and TNF-alpha secretion. The data herein demonstrate that DC can be infected by human C. trachomatis serovars and that chlamydial components regulate the secretion of various cytokines in DC. Collectively, these data suggest that DC play a role in the inflammatory processes caused by chlamydial infections.

Human CD8+ T cells recognize the 60-kDa cysteine-rich outer membrane protein from Chlamydia trachomatis.

The intracellular bacterial pathogen Chlamydia is sequestered from the host cell cytoplasm by remaining within an inclusion body during its replication cycle. Nevertheless, CD8(+) T cells recognizing Chlamydia Ags in the context of MHC class I molecules are primed during infection. We have recently described derivation of Chlamydia-specific human CD8(+) T cells by using infected dendritic cells as a surrogate system to reflect Chlamydia-specific CD8(+) T cell responses in vivo. These CD8(+) T cell clones recognize chlamydial Ags processed via the conventional class Ia processing pathway, as assessed by treatment of infected APC with lactacystin and brefeldin A, suggesting that the Ags are translocated from the chlamydial inclusion into the host cell cytosol. In this study, outer membrane protein 2 (OmcB) was identified as the Ag recognized by one of these Chlamydia-specific human CD8(+) T cells, and we defined the HLA*A0101-restricted epitope from this Ag. CD8(+) T cell responses to this epitope were present at high frequencies in the peripheral blood of both of two HLA*A0101 donors tested. In vitro chlamydial growth was completely inhibited by the OmcB-specific CD8(+) T cell clone independently of lytic mechanisms. OmcB is a 60-kDa protein that has been postulated to be associated with the Chlamydia outer membrane complex. The subcellular localization of OmcB to the cytosol of infected cells, as determined by conventional MHC class I Ag processing and presentation, suggests the possibility of an additional, cytosolic-associated function for this protein.

Functional characterization of class Ia- and non-class Ia-restricted Chlamydia-reactive CD8+ T cell responses in humans.

CD8(+) T cells are a key immune component for the eradication of many intracellular pathogens. This study aims to characterize the human CD8(+) T cell response to naturally processed chlamydial Ags in individuals exposed to the intracellular pathogen Chlamydia trachomatis. By using C. trachomatis-infected autologous dendritic cells (DCs) as stimulators, Chlamydia-reactive CD8(+) T cell responses were detected in all 10 individuals tested. The majority of the Chlamydia-reactive CD8(+) T cells were non-MHC class Ia restricted in all three of the individuals tested. From one donor, three non-class Ia-restricted and two class Ia-restricted Chlamydia-specific CD8(+) T cells were cloned and characterized further. All five T cell clones secreted IFN-gamma in response to autologous DCs infected with viable Chlamydia, but not with DCs pulsed with inactivated chlamydial elementary bodies. MHC class Ia-restricted and non-class Ia-restricted responses were inhibited by DC treatment with a proteasomal inhibitor and an endoplasmic reticulum-Golgi transport inhibitor, suggesting that these T cells recognize a peptide Ag translocated to the host cell cytosol during infection that is processed via the classical class Ia Ag-processing pathway. Even though both restricted and nonrestricted CD8(+) T cells produced IFN-gamma in response to Chlamydia-infected fibroblasts, only the non-class Ia-restricted cells were lytic for these targets. The class Ia-restricted CTLs, however, were capable of cytolysis as measured by redirected killing. Collectively, these data demonstrate that both class Ia-restricted and non-classically restricted CD8(+) T cells are elicited in C. trachomatis-exposed individuals. Their role in host immunity remains to be elucidated.