Non-specific effects (NSE) of vaccines such as BCG may protect against subsequent infections beyond their target pathogens, via both innate and adaptive immune mechanisms. We hypothesized that BCG vaccination would be associated with lower rates of common pediatric infections. We performed a secondary analysis of data collected during a placebo-controlled trial of BCG vaccination among American Indian/Alaska Native (AI/AN) schoolchildren, conducted from 1935–1938. This study was approved by the USUHS Institutional Review Board. The trial enrolled 2,860 AI/AN schoolchildren and prospectively recorded post-vaccination medical histories through 1947. Inclusion criteria were negative tuberculin skin test, normal chest radiograph, and receipt of study vaccine. Children with prior varicella, parotitis, measles, or pertussis were excluded. Outcomes included subsequent pneumonia/pleurisy, pertussis, measles, mumps, rubella, varicella, influenza, and infectious (non-TB) mortality. Cox proportional hazards models estimated hazard ratios (HRs) and 95% confidence intervals (CIs), adjusted for age, sex, and region. Kaplan-Meier time-to-event analyses were also performed. Among enrolled children, 1,489 received BCG and 1,371 placebo. Adjusted HRs (95% CI) were: non-TB infectious mortality 0.79 (0.54–1.47), pneumonia 0.84 (0.64–1.11), rubella 0.94 (0.64–1.37), varicella 0.95 (0.70–1.29), mumps 0.98 (0.80–1.19), influenza 1.01 (0.80–1.28), measles 1.06 (0.88–1.27), and pertussis 1.08 (0.85–1.37). Log-rank tests suggested differences between BCG and placebo for pneumonia (p=0.21), pertussis (p=0.57), measles (p=0.52), varicella (p=0.74), rubella (p=0.71), mumps (p=0.79), and influenza (p=0.92). Five of eight outcomes had HRs < 1.0, suggesting a trend towards protective effects. However, none reached statistical significance. BCG vaccination in school-aged children trended toward NSE protection but did not significantly reduce infection rates or infectious mortality in this pre-vaccine era population. Disclaimer: The views expressed in this abstract are those of the author(s) and do not necessarily reflect the official policy of the Department of Defense or the US government. Authors report no conflicts of interest. Naomi E. Aronson, MD, Wolters Kluwer: royalties for writing for uptodate

Tuberculosis (TB) leads global infectious diseases that cause mortality. Widely used BCG vaccine protects against TB disease (especially miliary, meningeal forms) but the protection of primary TB is inconclusive. Our objective was to assess the effectiveness of BCG vaccine to prevent primary TB using prospectively collected data in a clinical trial. This study was approved by the USUHS Institutional Review Board. Data from a controlled trial of BCG (Phipps) in American Indian/Alaskan Native (AI/AN) schoolchildren, n=2763, enrolled 1935-8, and a case control trial of 257 newborns in Rosebud SD, Belcourt ND enrolled 1939-40, were reviewed. Before (schoolchildren only) and after BCG vaccination annual intradermal PPD testing and chest radiographs (CXR) were performed through 1947. Inclusion criteria for this analysis were negative PPD and normal CXR prior to vaccination (schoolchildren), received placebo or BCG vaccine, had a follow-up assessment. Exclusion included reactive or undocumented PPD or abnormal CXR prior to immunization. Primary pulmonary TB was defined as hilar adenopathy, lower lobe infiltrates without other identified etiology, +/- pleural effusion. In the control arm, conversion of PPD >10mm was considered in the context of radiographic findings. Severe TB was miliary or meningeal. Cox proportional hazards regression model with hazard ratios (HR) and 95% confidence intervals (95%CI) was performed for outcomes of primary pulmonary TB and severe TB. 2731 AI/AN schoolchildren, mean age 7.6 years, were evaluated. 1416 received BCG, 1315 placebo. Primary pulmonary TB occurred in 122 BCG, 302 placebo recipients with a sex, age, region-adjusted Cox proportional HR of 0.34, 95% CI 0.28,0.42 for BCG vaccine. 253 newborns were analyzed; 116 received BCG, 137 placebo. For newborns a sex and region adjusted HR for any TB was reported.CohortPrimary TB adjusted HR (BCG)Severe TB adjusted HR (BCG)Schoolchildren n=27310.34 (95CI 0.28,0.42)0.24 (95CI 0.08, 0.74)Newborn n=2530.36 (95CI 0.13, 0.996)0 cases BCG, 2 cases placebo In this heavily TB-exposed AI/AN population, BCG was quite effective in preventing primary pulmonary TB in newborns (64%) or schoolchildren (66%), as well as meningeal or miliary TB (76%). Naomi E. Aronson, MD, Wolters Kluwer: royalties for writing for uptodate Lee Harrison, MD, GSK: Advisor/Consultant|Merck: Board Member|Pfizer: Advisor/Consultant|Sanofi: Advisor/Consultant

This paper will update readers on recent developments in leishmaniasis in the areas of epidemiology, diagnosis, treatment and prevention. As control of visceral leishmaniasis in India, Nepal and Bangladesh is heralded, East Africa becomes the region with most cases. Globally, cutaneous leishmaniasis rates continue to rise, with relocation and conflict in endemic areas contributory. In the US, molecular approaches to the diagnosis of leishmaniasis have become the primary tool for species determination. Some issues related to treatment of immunocompromised hosts are reviewed. An approach to human vaccines for leishmaniasis, with three vaccines in clinical development, is summarized. New incremental advances in both visceral and cutaneous leishmaniasis are identified. These span changes in epidemiology, identification of new species that infect humans, advances in laboratory confirmation, a transcriptomic approach to understanding changes in the skin in the initial weeks of infection, and management in immunocompromised hosts.

IntroductionCutaneous leishmaniasis is a neglected vector-borne parasitic disease prevalent in 92 countries with approximately one million new infections annually. Interactions between vector saliva and the human host alter the response to infection and outcome of disease.MethodsTo characterize the human immunological responses developed against saliva of Phlebotomus duboscqi, a Leishmania major (L. major) vector, we repeatedly exposed the arms of 14 healthy U.S volunteers to uninfected P. duboscqi bites. Blood was collected a week after each exposure and used to assess total IgG antibodies against the proteins of P. duboscqi salivary gland homogenate (SGH) and the levels of IFN-gamma and IL-10 from peripheral blood mononuclear cells (PBMCs) stimulated with SGH or recombinant sand fly proteins. We analyzed skin punch biopsies of the human volunteer arms from the insect bite site and control skin site after multiple P. duboscqi exposures (four volunteers) using immunohistochemical staining.ResultsA variety of immediate insect bite skin reactions were observed. Late skin reactions to insect bites were characterized by macular hyperpigmentation and/or erythematous papules. Hematoxylin and eosin staining showed moderate mononuclear skin infiltrate with eosinophils in those challenged recently (within 2 months), eosinophils were not seen in biopsies with recall challenge (6 month post bites). An increase in plasma antigen-specific IgG responses to SGH was observed over time. Western Blot results showed strong plasma reactivity to five P. duboscqi salivary proteins. Importantly, volunteers developed a cellular immunity characterized by the secretion of IFN-gamma upon PBMC stimulation with P. duboscqi SGH and recombinant antigens.DiscussionOur results demonstrate that humans mounted a local and systemic immune response against P. duboscqi salivary proteins. Specifically, PduM02/SP15-like and PduM73/adenosine deaminase recombinant salivary proteins triggered a Th1 type immune response that might be considered in future development of a potential Leishmania vaccine.

BACKGROUND:Tuberculosis vaccine trials using disease as the primary endpoint are large, time consuming, and expensive. An earlier immunological measure of the protection against disease would accelerate tuberculosis vaccine development. We aimed to assess whether the effectiveness of the Bacillus Calmette-Guérin (BCG) vaccine for prevention of Mycobacterium tuberculosis infection was consistent with that for prevention of tuberculosis disease. METHODS:We conducted an individual participant data (IPD) meta-analysis on experimental and observational longitudinal studies before April 6, 2018, identified through systematic reviews, known to us through expert knowledge in the field, reporting on BCG vaccination status, M tuberculosis infection test (QuantiFERON IFN-γ release assay [IGRA] and tuberculin skin test [TST]), and tuberculosis incidence. Cohort studies were included only for countries with a mandatory neonatal BCG vaccination policy. Exclusion criteria were previous or current tuberculosis disease, HIV infection, tuberculosis preventive treatment usage, and for household contacts, a positive baseline IGRA or TST test and young children aged 0-2 years; for randomised controlled trials, TST results within 2 years after random assignation were excluded. We contacted the investigators of the identified studies to provide IPD. We compared the protective efficacy of the BCG vaccine against M tuberculosis infection with that against tuberculosis disease using mixed-effects, multivariable proportional hazards modelling, by study type, M tuberculosis infection test (IGRA and TST), cutoff for defining test positivity, age, sex, and latitude. FINDINGS:We identified 79 studies eligible for full screening and of these, IPD datasets from 14 studies were included in our analysis: 11 household contact studies (29 147 participants), two adolescent cohort studies (11 368 participants), and one randomised controlled trial (2963 participants). Among 28 188 participants we found no protection by the BCG vaccine against TST conversion regardless of cutoff in any type of study. Among 1491 household contacts, but not among 5644 adolescents, the BCG vaccine protected against QuantiFERON conversion at the primary cutoff of 0·7 IU/mL or more with the adjusted hazard ratio (0·65, 95% CI 0·51-0·82) being consistent with that for protection against disease (0·68, 0·18-2·59). Protection against QuantiFERON conversion at cutoff of 0·35 IU/mL or more (0·64, 0·51-0·81) was similar. INTERPRETATION:Protection from the BCG vaccination against M tuberculosis infection, measured as QuantiFERON conversion, is inconsistent across different groups. Among groups with recent household exposure, QuantiFERON conversion is consistent with protection against disease and could be evaluated as a proxy for disease in tuberculosis vaccine trials. We found that TST lacks value for prevention in phase 2b proof-of-concept trials. FUNDING:Bill & Melinda Gates Foundation.

Abstract Background Bacille Calmette Guerin (BCG) vaccine’s protective effect against tuberculosis (TB) is established. BCG vaccine also has off-target effects, perhaps due to long-lasting trained immunity. BCG provides unanticipated beneficial effects against other infectious/non-infectious diseases, including respiratory infections, diabetes mellitus, dementia, and cancer. We aimed to determine if there was differential mortality over a lifetime in those receiving BCG versus placebo. We analyzed all cause and infectious diseases (ID) mortality in a longitudinal cohort study of American Indians and Alaska Natives (AI/AN) enrolled in a BCG efficacy clinical trial. Methods 2963 AI/AN participants were enrolled median age 7.6 years (range 0.1-20) in a saline placebo-controlled BCG trial between 1935-8 in five US geographic regions, with prospective follow up through 1948. Retrospective data collection was completed from 1992-8. Mortality data was supplemented by National Death Index search in 2006, pending 2023. Demographics, medical and mortality history were collected blinded to vaccine arm. Differences in mortality were analyzed by vaccine, demographic and death characteristics via bivariate association using chi-square, t-test and logistic regression with STATA. Results 1540 BCG, 1423 placebo participants enrolled. At 71 years from the trial start, 1600/2963 (54.0%) participants were deceased; 1171 (73.2%) to non-ID, 94 (5.9%) TB, 217 (13.6%) other infections, and 118 (7.3%) were unknown. Median age at death was similar (BCG 55.0 years, placebo 54.0). BCG vaccinated participants had a 31% decreased odds of death vs. placebo for ID not-TB (CI=0.52-0.93, p = 0.014) and 74% decreased odds of death due to TB (CI=0.16-0.42, p=.000). Adjusted logistic regression analysis showed BCG decreased odds of all cause death by 14% (p = 0.04). Male sex (OR 1.77) and living in Arizona (OR 2.53) or Wyoming (2.24) were associated with increased all cause mortality (p< 0.001). Conclusion In this cohort, BCG vaccine conferred a mortality benefit against TB but also other infections. Male sex and certain geographic regions were associated with higher odds of death. These findings suggest that childhood vaccination with BCG vaccine may reduce mortality beyond preventing TB deaths. Disclosures Lee Harrison, MD, GSK: Advisor/Consultant|Merck: Advisor/Consultant|Pfizer: Advisor/Consultant|Sanofi: Advisor/Consultant Naomi E. Aronson, MD, british medical journal: Honoraria|British Medical Journal: honoraria for writing chapter for Best Evidence|Elsevier: royallties serve as textbook editor|Elsevier: Royalties as text editor|UpTo Date: royalties for writing chapters|UpToDate: royalties for writing chapters|Wellcome Foundation: Honoraria|Wellcome Foundation: program advisory board|Wellcome Trust: Honoraria|Wellcome Trust: program advisory board Naomi E. Aronson, MD, british medical journal: Honoraria|British Medical Journal: honoraria for writing chapter for Best Evidence|Elsevier: royallties serve as textbook editor|Elsevier: Royalties as text editor|UpTo Date: royalties for writing chapters|UpToDate: royalties for writing chapters|Wellcome Foundation: Honoraria|Wellcome Foundation: program advisory board|Wellcome Trust: Honoraria|Wellcome Trust: program advisory board

Visceral leishmaniasis (VL) is a chronic infection caused by Leishmania (L.) donovani or L. infantum parasites. Despite having the infection, most individuals never develop the clinical disease and are able to control the parasite and remain asymptomatic. However, some progress to symptomatic VL, leading to death if untreated. The host immune response has a major role in determining the progression and severity of the clinical manifestations in VL; several immune biomarkers of symptomatic VL have been described with interferon-gamma release as a surrogate biomarker of host cellular immunity. However, new biomarkers to identify asymptomatic VL (AVL) are needed for the identification of people at risk for VL activation. In our study, levels of chemokine/cytokine in the supernatants of peripheral mononuclear blood cells (PBMC) from 35 AVL+ Iraq-deployed participants, stimulated in vitro with soluble Leishmania antigen for 72 h, were assessed by a bead-based assay that allows the measurement of multiple analytes. PBMC of AVL-negative military beneficiaries were used as controls. Monocyte Chemoattractant Protein-1, Monokine Induced by Gamma Interferon and Interleukin-8, were detected at high levels in AVL+ stimulated cultures from Iraq deployers compared to uninfected controls. Measurement of chemokine/cytokine levels can identify cellular immune responses in AVL+ asymptomatic individuals.

Abstract Background Visceral leishmaniasis (VL) is a chronic protozoal disease caused from the bite of an infected sand fly. VL due to Leishmania infantum is endemic in Afghanistan, although its distribution is poorly characterized. The spectrum of VL ranges from asymptomatic to active VL with symptoms of fever, weight loss, organomegaly, and pancytopenia. Most symptomatic patients die without treatment; asymptomatic patients remain at risk lifelong for symptomatic reactivation—especially if immunosuppressed. Travelers returning from VL endemic areas may harbor latent infection years after their return. We aimed to determine the prevalence of asymptomatic VL (AVL) in US military personnel previously deployed to Afghanistan. Methods Healthy adult US military personnel who deployed to Afghanistan over summer months were recruited from the Washington DC area. 90 volunteers completed a risk factor survey, blood draw, and had completed results. Diagnostic testing utilized Leishmania ELISA, interferon gamma release assay (IGRA), and quantitative PCR (qPCR). Statistical analyses included Fisher exact test, Pearson χ2 test, Welch Two Sample t-test, and Mann-Whitney U test. IRB approval was obtained. Results The prevalence of AVL (ELISA, IGRA, or qPCR positivity) in the volunteers was 9/90 (10%). Two (2.2%) PCR, 7 (7.8%) ELISA, and no (0%) IGRA samples were positive. AVL+ participants were a median of 10.6 years (range 8 - 18.6) post Afghanistan deployment, and compared to AVL- volunteers, were older (median 50 versus 40 years, p= 0.008) and more likely to self-identify as African-American (n = 5 (56%) vs 16 (20%), p = 0.029). No risk factors for AVL were identified in exploratory analysis of the volunteers’ military roles, types of outdoor exposure, or deployment location within Afghanistan. Conclusion In this preliminary cross-sectional analysis of US military personnel who returned from Afghanistan, the prevalence of AVL was 10%–comparable to other published studies on the prevalence of AVL in endemic areas and of concern when one considers the several million US military personnel previously deployed to Iraq and Afghanistan. Due to the persistence of this intracellular infection, clinicians should be alerted for reactivation potential in previously deployed Servicemembers. Disclosures Naomi E. Aronson, MD, british medical journal: Honoraria|British Medical Journal: honoraria for writing chapter for Best Evidence|Elsevier: royallties serve as textbook editor|Elsevier: Royalties as text editor|UpTo Date: royalties for writing chapters|UpToDate: royalties for writing chapters|Wellcome Foundation: Honoraria|Wellcome Foundation: program advisory board|Wellcome Trust: Honoraria|Wellcome Trust: program advisory board Naomi E. Aronson, MD, british medical journal: Honoraria|British Medical Journal: honoraria for writing chapter for Best Evidence|Elsevier: royallties serve as textbook editor|Elsevier: Royalties as text editor|UpTo Date: royalties for writing chapters|UpToDate: royalties for writing chapters|Wellcome Foundation: Honoraria|Wellcome Foundation: program advisory board|Wellcome Trust: Honoraria|Wellcome Trust: program advisory board

The advent of combined antiretroviral therapy (cART) has been instrumental in controlling HIV-1 replication and transmission and decreasing associated morbidity and mortality. However, cART alone is not able to cure HIV-1 due to the presence of long-lived, latently infected immune cells, which re-seed plasma viremia when cART is interrupted. Assessment of HIV-cure strategies using ex vivo culture methods for further understanding of the diversity of reactivated HIV, viral outgrowth, and replication dynamics are enhanced using ultrasensitive digital ELISA based on single-molecule array (Simoa) technology to increase the sensitivity of endpoint detection. In viral outgrowth assays (VOA), exponential HIV-1 outgrowth has been shown to be dependent upon initial virus burst size surpassing a critical growth threshold of 5100 HIV-1 RNA copies. Here, we show an association between ultrasensitive HIV-1 Gag p24 concentrations and HIV-1 RNA copy number that characterize viral dynamics below the exponential replication threshold. Single-genome sequencing (SGS) revealed the presence of multiple identical HIV-1 sequences, indicative of low-level replication occurring below the threshold of exponential outgrowth early during a VOA. However, SGS further revealed diverse related HIV variants detectable by ultrasensitive methods that failed to establish exponential outgrowth. Overall, our data suggest that viral outgrowth occurring below the threshold necessary for establishing exponential growth in culture does not preclude replication competence of reactivated HIV, and ultrasensitive detection of HIV-1 p24 may provide a method to detect previously unquantifiable variants. These data strongly support the use of the Simoa platform in a multi-prong approach to measuring latent viral burden and efficacy of therapeutic interventions aimed at an HIV-1 cure.

Leishmania major , transmitted in Iraq by the bite of a sand fly Phlebotomus papatasi , causes cutaneous leishmaniasis (CL). The sand fly saliva is immunogenic, with both systemic humoral and cellular human immune responses resulting from natural exposure. 248 Americans who developed L. major infection in Iraq were sex, race/ethnicity, year of Iraq deployment-matched to controls without CL. Using a case-control study design, we compared sand fly saliva-specific human IgG levels and recognized antigens between the two groups. Serologic responses to Ph. papatasi salivary gland homogenate were studied with ELISA and Western blot, using serial samples obtained from before travel, during CL treatment (CL) or at time of return to US (controls), as well as (for CL cases) six to 24 months after return to non-endemic US. The mean change in optical density (MCOD), reflecting the change in sand fly saliva-specific IgG before and after exposure in Iraq, was 0.296 (range -0.138 to 2.057) in cases and 0.151 (range -0.454 to1.085) in controls, p<0.001. Low levels of sand fly saliva specific antibody were noted in CL cases by 7-8 months after return to the US. The most frequently recognized Ph. papatasi salivary antigens were MW30 (PpSP32) and MW64, although other salivary proteins recognized were MW12/14, 15, 18, 28, 32, 36, 42, 44, 46, 52. Logistic regression suggested that MW15, 28 and 42 were associated with the largest effect on the MCOD. MW30 was the most frequently recognized antigen suggesting a role as biomarker for sand fly exposure and CL risk. Anti- Ph . papatasi saliva IgG waned within months of return to the US. We also discuss vector antigenic saliva proteins in the context of CL presentation and identify some salivary antigens that may correlate with less lesion area, ulcer versus papule/plaque, race among those with CL.

Abstract Background Cutaneous leishmaniasis (CL) is a threat to U.S. Military personnel as they deploy to endemic areas. As treatment may require evacuation, CL undermines operations. Elucidating the epidemiology of CL in this population is key for prevention. Methods We retrospectively reviewed data from a CL sodium stibogluconate treatment trial at Walter Reed Army Medical Center, Washington DC. 412 military members with parasitologically confirmed CL and deployment to Southwest Asia from May 2002 - August 2004 enrolled. Subjects’ CL lesions were counted and measured. 334 subjects completed a risk survey. Given no control group, we used number of CL lesions (NL), total lesion area (TLA), and lesion location as outcomes to assess CL risks. Non-parametric tests and logistic regression were used as appropriate. Results Permethrin treated bed net use was associated with lower NL (p = 0.000), TLA (p = 0.024), and odds of head/face lesion (OR 0.12, p = 0.047). Sleeping in a combat uniform was associated with lower TLA (p = 0.000) and odds of leg/foot lesion (OR 0.39, p = 0.023). Use of a permethrin treated uniform (p = 0.002) and N,N-diethyl-meta-toluamide (DEET) insect repellent at night (p = 0.046) were associated with lower NL. Noting unit members with similar lesions was associated with higher NL (p = 0.007) and odds of head/face lesion (OR 12.1, p = 0.019). National Guard status followed by Active Duty was associated with higher NL (p = 0.002) and TLA (p = 0.000) than Reserve. Sleeping in a building was associated with higher TLA (p = 0.008) and odds of arm lesion (OR 2.28, p = 0.016). Sleeping near animal burrows was associated with higher NL (p = 0.031). Sleeping on a cot (p = 0.006), certain ethnicities (p = 0.020) and certain military occupational specialties (p =0.038) were associated with higher TLA. Increasing age (p = 0.001) and years of service (p = 0.000) were positively correlated with TLA. Conclusion This is the largest group of U.S. Military members with CL reported and provides insight into risks for CL which will guide preventive efforts to reduce the burden of illness in this population. The opinions and assertions expressed herein are those of the author(s) and do not reflect the official policy or position of the Uniformed Services University of the Health Sciences, the Department of Defense, or the U.S. Government. Disclosures Naomi E. Aronson, M.D., Elsevier: Royalties as text editor, honoraria for chapter writing|UpToDate: royalties for writing chapters.

Incidence of visceral leishmaniasis (VL) in the Indian subcontinent (ISC) has declined by more than 95% since initiation of the elimination program in 2005. As the ISC transitions to the postelimination surveillance phase, an accurate measurement of human-vector contact is needed to assure long-term success. To develop this tool, we identified PagSP02 and PagSP06 from saliva of Phlebotomus argentipes, the vector of Leishmania donovani in the ISC, as immunodominant proteins in humans. We also established the absence of cross-reactivity with Phlebotomus papatasi saliva, the only other human-biting sand fly in the ISC. Importantly, by combining recombinant rPagSP02 and rPagSP06 we achieved greater antibody recognition and specificity than single salivary proteins. The receiver operating characteristics curve for rPagSP02 + rPagSP06 predicts exposure to Ph. argentipes bites with 90% specificity and 87% sensitivity compared to negative control sera (P >.0001). Overall, rPagSP02 + rPagSP06 provides an effective surveillance tool for monitoring vector control efforts after VL elimination.

Leishmaniasis, a chronic and persistent intracellular protozoal infection caused by many different species within the genus Leishmania, is an unfamiliar disease to most North American providers. Clinical presentations may include asymptomatic and symptomatic visceral leishmaniasis (so-called Kala-azar), as well as cutaneous or mucosal disease. Although cutaneous leishmaniasis (caused by Leishmania mexicana in the United States) is endemic in some southwest states, other causes for concern include reactivation of imported visceral leishmaniasis remotely in time from the initial infection, and the possible long-term complications of chronic inflammation from asymptomatic infection. Climate change, the identification of competent vectors and reservoirs, a highly mobile populace, significant population groups with proven exposure history, HIV, and widespread use of immunosuppressive medications and organ transplant all create the potential for increased frequency of leishmaniasis in the U.S. Together, these factors could contribute to leishmaniasis emerging as a health threat in the U.S., including the possibility of sustained autochthonous spread of newly introduced visceral disease. We summarize recent data examining the epidemiology and major risk factors for acquisition of cutaneous and visceral leishmaniasis, with a special focus on implications for the United States, as well as discuss key emerging issues affecting the management of visceral leishmaniasis.

Background Sand fly saliva exposure plays an important role in immunity against leishmaniasis where it has mostly been associated with protection. Phlebotomus (Ph . ) alexandri transmits Leishmania (L . ) infantum , the causative agent of visceral leishmaniasis (VL), in Iraq. Our group recently demonstrated that 20% of Operation Iraqi Freedom (OIF) deployers had asymptomatic VL (AVL) indicative of prior infection by the parasite L . infantum . Little is known about Ph . alexandri saliva, and the human immune response to it has never been investigated. Here, we characterize the humoral and cellular immune response to vector saliva in OIF deployers naturally exposed to bites of Ph . alexandri and characterize their immunological profiles in association to AVL. Methodology/Principal findings The humoral response to Ph . alexandri salivary gland homogenate (SGH) showed that 64% of 200 OIF deployers developed an antibody response. To assess the cellular immune response to saliva, we selected a subcohort of subjects based on their post-travel (median 4 months; range 1–22 months) antibody response (SGH Antibody [Ab] positive or negative) as well as their AVL status; ten never-traveled controls were also included. Banked peripheral blood mononuclear cells (PBMC), collected ~10 years after end of deployment, were stimulated with SGH for 96 hours. The levels of IFN- γ, IL-6, IL-10, IL-13 and IL-17 were determined by ELISA. Our findings indicate that OIF deployers mounted a cellular response to SGH where the anti-SGH+ asymptomatic subjects developed the highest cytokine levels. Further, stimulation with SGH produced a mixture of pro-inflammatory and anti-inflammatory cytokines. Contrary to our hypothesis, we observed no correlation between the cellular immune response to Ph . alexandri SGH and prevention from asymptomatic infection with L . infantum . Conclusions/Significance As we found, although all infected deployers demonstrated persistent disease control years after deployment, this did not correlate with anti-saliva systemic cellular response. More exposure to this vector may facilitate transmission of the L . infantum parasite. Since exposure to saliva of Ph . alexandri may alter the human immune response to bites of this vector, this parameter should be taken into consideration when considering the VL risk.

Background. Dengue is a global health problem and the development of a tetravalent dengue vaccine with durable protection is a high priority. A heterologous prime-boost strategy has the advantage of eliciting immune responses through different mechanisms and therefore may be superior to homologous prime-boost strategies for generating durable tetravalent immunity. Methods. In this phase 1 first-in-human heterologous prime-boost study, 80 volunteers were assigned to 4 groups and received a tetravalent dengue virus (DENV-1-4) purified inactivated vaccine (TDENV-PIV) with alum adjuvant and a tetravalent dengue virus (DENV-1-4) live attenuated vaccine (TDENV-LAV) in different orders and dosing schedules (28 or 180 days apart). Results. All vaccination regimens had acceptable safety profiles and there were no vaccine-related serious adverse events. TDEN-PIV followed by TDEN-LAV induced higher neutralizing antibody titers and a higher rate of tetravalent seroconversions compared to TDEN-LAV followed by TDEN-PIV. Both TDEN-PIV followed by TDEN-LAV groups demonstrated 100% tetravalent seroconversion 28 days following the booster dose, which was maintained for most of these subjects through the day 180 measurement. Conclusions. A heterologous prime-boost vaccination strategy for dengue merits additional evaluation for safety, immunogenicity, and potential for clinical benefit.