Development and validation of RT-LAMP for detecting yellow fever virus in non-human primates samples from Brazil.

Monitoring yellow fever in non-human primates (NHPs) is an early warning system for sylvatic yellow fever outbreaks, aiding in preventing human cases. However, current diagnostic tests for this disease, primarily relying on RT-qPCR, are complex and costly. Therefore, there is a critical need for simpler and more cost-effective methods to detect yellow fever virus (YFV) infection in NHPs, enabling early identification of viral circulation. In this study, an RT-LAMP assay for detecting YFV in NHP samples was developed and validated. Two sets of RT-LAMP primers targeting the YFV NS5 and E genes were designed and tested together with a third primer set to the NS1 locus using NHP tissue samples from Southern Brazil. The results were visualized by colorimetry and compared to the RT-qPCR test. Standardization and validation of the RT-LAMP assay demonstrated 100% sensitivity and specificity compared to RT-qPCR, with a detection limit of 12 PFU/mL. Additionally, the cross-reactivity test with other flaviviruses confirmed a specificity of 100%. Our newly developed RT-LAMP diagnostic test for YFV in NHP samples will significantly contribute to yellow fever monitoring efforts, providing a simpler and more accessible method for viral early detection. This advancement holds promise for enhancing surveillance and ultimately preventing the spread of yellow fever.

Mitochondrial Hyperactivity and Reactive Oxygen Species Drive Innate Immunity to the Yellow Fever Virus-17D Live-Attenuated Vaccine.

The yellow fever virus 17D (YFV-17D) live attenuated vaccine is considered one of the successful vaccines ever generated associated with high antiviral immunity, yet the signaling mechanisms that drive the response in infected cells are not understood. Here, we provide a molecular understanding of how metabolic stress and innate immune responses are linked to drive type I IFN expression in response to YFV-17D infection. Comparison of YFV-17D replication with its parental virus, YFV-Asibi, and a related dengue virus revealed that IFN expression requires RIG-I-like Receptor signaling through MAVS, as expected. However, YFV-17D uniquely induces mitochondrial respiration and major metabolic perturbations, including hyperactivation of electron transport to fuel ATP synthase. Mitochondrial hyperactivity generates reactive oxygen species (mROS) and peroxynitrite, blocking of which abrogated IFN expression in non-immune cells without reducing YFV-17D replication. Scavenging ROS in YFV-17D-infected human dendritic cells increased cell viability yet globally prevented expression of IFN signaling pathways. Thus, adaptation of YFV-17D for high growth uniquely imparts mitochondrial hyperactivity generating mROS and peroxynitrite as the critical messengers that convert a blunted IFN response into maximal activation of innate immunity essential for vaccine effectiveness.

Next generation yellow fever vaccine induces an equivalent immune and transcriptomic profile to the current vaccine: observations from a phase I randomised clinical trial.

BACKGROUND: Yellow fever (YF), a mosquito-borne acute viral haemorrhagic illness, is endemic to many tropical and subtropical areas of Africa and Central and South America. Vaccination remains the most effective prevention strategy; however, as repeated outbreaks have exhausted vaccine stockpiles, there is a need for improved YF vaccines to meet global demand. A live-attenuated YF vaccine candidate (referred to as vYF) cloned from a YF-17D vaccine (YF-VAX®) sub-strain, adapted for growth in Vero cells cultured in serum-free media, is in clinical development. We report the innate and adaptive immune responses and the transcriptome profile of selected genes induced by vYF. METHODS: Healthy adults aged 18-60 years were randomised at a 1:1:1:1 ratio to receive one dose of vYF at 4, 5 or 6 Log CCID50 or YF-VAX (reference vaccine), administered subcutaneously in the upper arm (ClinicalTrials.gov identifier: NCT04142086). Blood/serum samples were obtained at scheduled time points through 180 days (D180) post-vaccination. The surrogate endpoints assessed were: serum cytokine/chemokine concentrations, measured by bead-based Multiplex assay; peripheral blood vYF-specific IgG and IgM memory B cell frequencies, measured by FluoroSpot assay; and expression of genes involved in the immune response to YF-17D vaccination by RT-qPCR. FINDINGS: There was no increase in any of the cytokine/chemokine concentrations assessed through D14 following vaccination with vYF or YF-VAX, except for a slight increase in IP-10 (CXCL10) levels. The gene expression profiles and kinetics following vaccination with vYF and YF-VAX were similar, inclusive of innate (antiviral responses [type-1 interferon, IFN signal transduction; interferon-stimulated genes], activated dendritic cells, viral sensing pattern recognition receptors) and adaptive (cell division in stimulated CD4+ T cells, B cell and antibody) immune signatures, which peaked at D7 and D14, respectively. Increases in vYF-specific IgG and IgM memory B cell frequencies at D28 and D180 were similar across the study groups. INTERPRETATION: vYF-induced strong innate and adaptive immune responses comparable to those induced by YF-VAX, with similar transcriptomic and kinetic profiles observed. FUNDING: Sanofi.

Insect cell production of chimeric virus-like particles based on human immunodeficiency virus GAG proteins and yellow fever virus envelope protein.

The yellow fever virus (YFV) is a single stranded RNA virus belonging to the genus Orthoflavivirus that is capable of zoonotic transmissions that infect nonhuman and human primates. It is endemic in Brazil with recurrent epidemics of the disease, and it is transmitted through mosquitoes. The detection and immunization against YFV and other flaviviruses are fundamental for the management of the impacts of the disease in human environments. In an ongoing effort to develop new approaches for diagnostics and immunizations, we expressed VLPs displaying the yellow fever virus envelope protein (YFE) using recombinant baculovirus in insect cells. By co-expressing HIV-1 Pr55Gag protein (GAG) together with YFE we were able to generate chimeric VLPs containing a GAG core together with an envelope containing the YFE protein. The YFE and the chimeric GAG-YFE VLPs have potential as vaccine candidates and as reagents for serological assays in the detection of these viruses in human sera.

Which Stranger's Disease? Immigration, Immunization, and the Whitening of Cuba in the Age of Atlantic Revolutions.

In 1804, Cuban physician Tomás Romay tried and failed to create the first yellow fever vaccine. The article analyzes his experimental efforts, foregrounding the enslaved and enlisted subjects at the center of this early vaccine trial. Though a scientific failure, this brief experiment, the desires and logics embedded within it, and the measures deployed in its wake - in the form of European whitening campaigns - allow us to consider the political uses of immunity during the Age of Atlantic Revolutions. Historicizing these events within the wider geopolitics of the Caribbean, the article explicates the central role that yellow fever immunization played in Cuban authorities' attempts to shore up their political and economic sovereignty in the midst of anti-colonial and anti-slavery resistance. As such, it shows how yellow fever and its threat to social and economic order fits within a broader history of vaccination as a mechanism of colonial governance. Finally, by situating Cuban efforts to prevent yellow fever alongside the health concerns of enslaved people - concerns that arguably informed their resistance to slavery - the article also demonstrates how ideas about immunity and political belonging increasingly intersected through whiteness as an elite ideal in the era that Cuba first became a slave society.

Immunogenicity and Protective Efficacy of Aerosolized Live-Attenuated Yellow Fever 17D Vaccine in Mice.

Yellow fever (YF), caused by the yellow fever virus (YFV), continually spreads and causes epidemics worldwide, posing a great threat to human health. The live-attenuated YF 17D vaccine (YF-17D) has been licensed for preventing YFV infection and administrated via the intramuscular (i.m.) route. In this study, we sought to determine the immunogenicity and protective efficacy of aerosolized YF-17D via the intratracheal (i.t.) route in mice. YF-17D stocks in liquids were successfully aerosolized into particles of 6 µm. Further in vitro phenotype results showed the aerosolization process did not abolish the infectivity of YF-17D. Meanwhile, a single i.t. immunization with aerosolized YF-17D induced robust humoral and cellular immune responses in A129 mice, which is comparable to that received i.p. immunization. Notably, the aerosolized YF-17D also triggered specific secretory IgA (SIgA) production in bronchoalveolar lavage. Additionally, all immunized animals survived a lethal dose of YFV challenge in mice. In conclusion, our results support further development of aerosolized YF-17D in the future.

The global incidence and trends of three common flavivirus infections (Dengue, yellow fever, and Zika) from 2011 to 2021.

Background: Flavivirus pose a continued threat to global health, yet their worldwide burden and trends remain poorly quantified. We aimed to evaluate the global, regional, and national incidence of three common flavivirus infections (Dengue, yellow fever, and Zika) from 2011 to 2021. Methods: Data on the number and rate of incidence for the three common flavivirus infection in 204 countries and territories were retrieved from the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2021. The estimated annual percent change (EAPC) was calculated to quantify the temporal trend during 2011-2016, 2016-2019, and 2019-2021, respectively. Results: In 2021, an estimated 59,220,428 individuals were infected globally, comprising 58,964,185 cases of dengue, 86,509 cases of yellow fever, and 169,734 cases of Zika virus infection. The age-standardized incidence rate (ASIR) of the three common flavivirus infections increased by an annual average of 5.08% (95% CI 4.12 to 6.05) globally from 2011 to 2016, whereas decreased by an annual average of -8.37% (95% CI -12.46 to -4.08) per year between 2016 to 2019. The ASIR remained stable during 2019-2021, with an average change of 0.69% (95% CI -0.96 to 2.37) per year globally for the three common flavivirus infections. Regionally, the burden of the three common flavivirus infections was primarily concentrated in those regions with middle income, such as South Asia, Southeast Asia, and Tropical Latin America. Additionally, at the country level, there was an inverted "U" relationship between the SDI level and the ASI. Notably, an increase in the average age of infected cases has been observed worldwide, particularly in higher-income regions. Conclusion: Flavivirus infections are an expanding public health concern worldwide, with considerable regional and demographic variation in the incidence. Policymakers and healthcare providers must stay vigilant regarding the impact of COVID-19 and other environmental factors on the risk of flavivirus infection and be prepared for potential future outbreaks.

Adverse events following immunization (AEFI) with fractional one-fifth and one-half doses of yellow fever vaccine compared to full dose in children 9-23 months old in Uganda, 2019-2020 - Preliminary report.

BACKGROUND: In 2016, the World Health Organization recommended that a fractional dose of yellow fever (YF) vaccine could be used in persons 2 years of age or older in response to an emergency that resulted in a global shortage of available YF vaccine. However, this recommendation did not extend to the youngest age group licensed for YF vaccine because there were no published data on the use or safety of fractional dose YF vaccination in children aged 9-23 months. We conducted a single-blind randomized controlled trial, comparing the immunogenicity and safety of fractional one-fifth and one-half doses of Bio-Manguinhos 17DD YF vaccine with full dose in children aged 9-23 months old in Uganda. In this paper, we present the interim analysis on safety. METHODS: Children aged 9-23 months presenting for routine well-child services were recruited for inclusion at one of three study sites. We collected data during March 26, 2019-August 31, 2020, on all adverse events following immunization (AEFI) during active surveillance for 28 days post-vaccination using multiple collection tools including a diary card with an objective measurement of fever. An independent team from the Uganda national AEFI Committee investigated and classified serious AEFI (SAE) according to Brighton Collaboration Criteria. RESULTS: Among 1053 enrolled children, 672 (64%) were reported to have a non-serious AEFI (NSAE) and 17 (2%) were reported to have a SAE. The most common AEFI were diarrhoea, fever, and rash, each reported by 355 (34%), 338 (33%), and 188 (18%) participants, respectively. Among 17 participants with SAE, eight were reported to have had seizures and five were hospitalised for seizures or other causes (respiratory symptoms, gastrointestinal illness, malaria). Four SAEs (deaths) occurred >28 days after vaccination. There were no reported cases of pre-specified or vaccine-related SAEs. We observed no significant difference in frequency or severity of adverse events among the study groups. CONCLUSIONS: Using comprehensive active surveillance monitoring, we did not identify any unexpected safety concerns among children aged <2 years receiving YF vaccination, including with the fractional doses. Although we identified a high number of both serious and non-serious AEFI, none were determined to be causally related to YF vaccination. These results provide evidence for the safety of fractional dose YF vaccination among children aged 9-23 months.

Yellow fever vaccine coverage and associated factors among under-five children in Kenya: Data from Kenyan Demographic and Health Survey 2022.

Yellow fever is a vaccine preventable hemorrhagic disease that leads to morbidity and mortality in the affected individuals. The only options for preventing and controlling its spread are through vaccination. Therefore, this study was conducted to estimate yellow fever vaccination coverage and associated factors among under-five children in Kenya. The total weighted samples of 2,844 children aged under-five were included in this study. The data were taken from the Kenyan Demographic and Health Survey 2022. In the multivariable analysis, the adjusted odds ratio with a 95% CI was used to declare significant associations of yellow fever vaccine. The yellow fever vaccine coverage among children aged under-five in Kenya was 18.50%. The significant factors associated with yellow fever vaccine coverage were: the age of the child older than 24 months (AOR = 1.7; 95% CI (1.17-2.58)); higher odds of yellow fever vaccination coverage was observed among older children, place of residence (AOR = 1.76; 95% CI (1.04-2.97)); higher odds was observed among urban residents, maternal education; primary education (AOR = 1.99; 95% CI (1.04-2.97)), secondary education (AOR = 2.85; 95% CI (1.41-5.76)), mothers who attended primary or secondary education have higher odds of yellow fever vaccination coverage, wealth index (AOR = 2.38; 95% CI (1.15-4.91)); higher odds of vaccination coverage was observed among poor households. Yellow fever vaccine coverage among under-five children in Kenya was low and has become an important public health concern. Policymakers and other stakeholders are recommended to focus on vaccination programs to prevent yellow fever disease.

No Yellow Fever Vaccine Reactions in IgE-Mediated Egg Allergic Patients.

INTRODUCTION: Egg allergy usually manifests during the initial 2 years of life, a period in which most vaccinations are administered. This often leads to delays in the application of some vaccines in patients with egg allergies, exposing them to a risk of contracting preventable infections. The aim of the study was to describe the frequency of reactions after applying the yellow fever vaccine (YFV) within a population with egg allergy. METHODS: This was a cohort study with retrospective, multicenter data (2014-2023). Patient records diagnosed with egg allergy were gathered from their initial egg-related reactions until their YFV administration. Information was also collected about hypersensitivity tests conducted for egg and YFV such as the skin prick test (SPT) and intradermal test (IDT). RESULTS: Among the 171 records analyzed, 23.9% of patients had a history of egg anaphylaxis. Out of these, 5 patients had a positive SPT and 21 IDT with the YFV. All patients tolerated the application of YFV without developing hypersensitivity reactions, regardless of the results of the YFV tests, the severity of egg reactions, the number of egg reactions, or the time since the last egg reaction. Out of the total patient cohort, 46.1% (79 individuals) encountered delays in receiving the YFV, and in this subset, 14% faced delays lasting longer than 12 months. CONCLUSION: The risk of allergic reactions with the YFV remains low. YFV tests generate delays in the vaccine application without providing high diagnostic accuracy. YFV should not be deferred even in patients with a history of severe egg reactions.

Development of a hydrogen peroxide-inactivated vaccine that protects against viscerotropic yellow fever in a non-human primate model.

Yellow fever virus (YFV) is endemic in >40 countries and causes viscerotropic disease with up to 20%-60% mortality. Successful live-attenuated yellow fever (YF) vaccines were developed in the mid-1930s, but their use is restricted or formally contraindicated in vulnerable populations including infants, the elderly, and people with compromised immune systems. In these studies, we describe the development of a next-generation hydrogen peroxide-inactivated YF vaccine and determine immune correlates of protection based on log neutralizing index (LNI) and neutralizing titer-50% (NT50) studies. In addition, we compare neutralizing antibody responses and protective efficacy of hydrogen peroxide-inactivated YF vaccine candidates to live-attenuated YFV-17D (YF-VAX) in a rhesus macaque model of viscerotropic YF. Our results indicate that an optimized, inactivated YF vaccine elicits protective antibody responses that prevent viral dissemination and lethal infection in rhesus macaques and may be a suitable alternative for vaccinating vulnerable populations who are not eligible to receive replicating live-attenuated YF vaccines.

Boric acid toxic sugar bait suppresses male Aedes aegypti (Diptera: Culicidae): wing beat frequency and amplitude, flight activity, fecundity, insemination, and mate-finding Allee effect.

BACKGROUND: Controlling the spread of arboviral diseases remains a considerable challenge due to the rapid development of insecticide resistance in Aedes mosquitoes. This study evaluated the effects of boric acid-containing toxic sugar bait (TSB) on field populations of resistant Aedes aegypti mosquitoes. In addition, this study examined the flight activity and wing beat frequency and amplitude of males and the flight activity, fecundity, and insemination of females after pairing with males exposed to TSB. The population dynamics of Aedes mosquitoes under imbalanced sex ratios were examined to simulate realistic field conditions for male suppression under the effect of TSB. RESULTS: The mortality of male mosquitoes was consistently high within 24 h after exposure. By contrast, the mortality of female mosquitoes was inconsistent, with over 70% mortality observed at 168 h. The flight activity and wing beat amplitude of treated males were significantly lower than those of controls, but no significant difference in wing beat frequency was detected. The fecundity and insemination of treated female mosquitoes were lower than those of controls. A simulation study indicated that considerably low male population densities led to mating failures, triggering a mate-finding Allee effect and resulting in persistently low population levels. CONCLUSION: Boric acid-containing TSB could effectively complement current chemical intervention approaches to control resistant mosquito populations. TSB is effective in reducing field male populations and impairing male flight activity and female-seeking behavior, resulting in decreased fecundity and insemination. Male suppression due to TSB potentially results in a small mosquito population. © 2024 Society of Chemical Industry.

Evaluation of the sentinel yellow fever surveillance system in Uganda, 2017-2022: strengths and weaknesses.

BACKGROUND: Uganda has a sentinel surveillance system in seven high-risk sites to monitor yellow fever (YF) patterns and detect outbreaks. We evaluated the performance of this system from 2017 to 2022. METHODS: We evaluated selected attributes, including timeliness (lags between different critical time points), external completeness (proportion of expected sentinel sites reporting ≥ 1 suspect case in the system annually), and internal completeness (proportion of reports with the minimum required data elements filled), using secondary data in the YF surveillance database from January 2017-July 2022. We conducted key informant interviews with stakeholders at health facility and national level to assess usefulness, flexibility, simplicity, and acceptability of the surveillance system. RESULTS: In total, 3,073 suspected and 15 confirmed YF cases were reported. The median time lag from sample collection to laboratory shipment was 37 days (IQR:21-54). External completeness was 76%; internal completeness was 65%. Stakeholders felt that the surveillance system was simple and acceptable, but were uncertain about flexibility. Most (71%) YF cases in previous outbreaks were detected through the sentinel surveillance system; data were used to inform interventions such as intensified YF vaccination. CONCLUSION: The YF sentinel surveillance system was useful in detecting outbreaks and informing public health action. Delays in case confirmation and incomplete data compromised its overall effectiveness and efficiency.

Levels of Angiopoietin 2 Are Predictive for Mortality in Patients Infected With Yellow Fever Virus.

In 2018 there was a large yellow fever outbreak in São Paulo, Brazil, with a high fatality rate. Yellow fever virus can cause, among other symptoms, hemorrhage and disseminated intravascular coagulation, indicating a role for endothelial cells in disease pathogenesis. Here, we conducted a case-control study and measured markers related to endothelial damage in plasma and its association with mortality. We found that angiopoietin 2 is strongly associated with a fatal outcome and could serve as a predictive marker for mortality. This could be used to monitor severe cases and provide care to improve disease outcome.

Risk of arboviral transmission and insecticide resistance status of Aedes mosquitoes during a yellow fever outbreak in Ghana.

BACKGROUND: In late 2021, Ghana was hit by a Yellow Fever outbreak that started in two districts in the Savannah region and spread to several other Districts in three regions. Yellow fever is endemic in Ghana. However, there is currently no structured vector control programme for Aedes the arboviral vector in Ghana. Knowledge of Aedes bionomics and insecticide susceptibility status is important to control the vectors. This study therefore sought to determine Aedes vector bionomics and their insecticide resistance status during a yellow fever outbreak. METHODS: The study was performed in two yellow fever outbreak sites (Wenchi, Larabanga) and two non-outbreak sites (Kpalsogu, Pagaza) in Ghana. Immature Aedes mosquitoes were sampled from water-holding containers in and around human habitations. The risk of disease transmission was determined in each site using stegomyia indices. Adult Aedes mosquitoes were sampled using Biogents Sentinel (BG) traps, Human Landing Catch (HLC), and Prokopack (PPK) aspirators. Phenotypic resistance to permethrin, deltamethrin and pirimiphos-methyl was determined with WHO susceptibility tests using Aedes mosquitoes collected as larvae and reared into adults. Knockdown resistance (kdr) mutations were detected using allele-specific multiplex PCR. RESULTS: Among the 2,664 immature Aedes sampled, more than 60% were found in car tyres. Larabanga, an outbreak site, was classified as a high-risk zone for the Yellow Fever outbreak (BI: 84%, CI: 26.4%). Out of 1,507 adult Aedes mosquitoes collected, Aedes aegypti was the predominant vector species (92%). A significantly high abundance of Aedes mosquitoes was observed during the dry season (61.2%) and outdoors (60.6%) (P < 0.001). Moderate to high resistance to deltamethrin was observed in all sites (33.75% to 70%). Moderate resistance to pirimiphos-methyl (65%) was observed in Kpalsogu. Aedes mosquitoes from Larabanga were susceptible (98%) to permethrin. The F1534C kdr, V1016I kdr and V410 kdr alleles were present in all the sites with frequencies between (0.05-0.92). The outbreak sites had significantly higher allele frequencies of F1534C and V1016I respectively compared to non-outbreak sites (P < 0.001). CONCLUSION: This study indicates that Aedes mosquitoes in Ghana pose a significant risk to public health. Hence there is a need to continue monitoring these vectors to develop an effective control strategy.

Baculovirus surface display of a chimeric E-NS1 protein of YFV protects against YFV infection.

Yellow fever (YF) is a disease caused by the homonymous flavivirus that can be prevented by a vaccine containing attenuated viruses. Since some individuals cannot receive this vaccine, the development of alternatives is desirable. Here, we developed a recombinant baculovirus (rBV) surface display platform utilizing a chimeric E-NS1 protein as a vaccine candidate. A pBacPAK9 vector containing the baculoviral GP64 signal peptide, the YFV prM, E, NS1 and the ectodomain of VSV-G sequences was synthesized. This transfer plasmid and the bAcGOZA bacmid were cotransfected into Sf9 cells, and an rBV-E-NS1 was obtained, which was characterized by PCR, WB, IFI and FACS analysis. Mice immunized with rBV-E-NS1 elicited a specific humoral and cellular immune response and were protected after YFV infection. In summary, we have developed an rBV that expresses YFV major antigen proteins on its surface, which opens new alternatives that can be tested in a mouse model.

Duration of post-vaccination immunity to yellow fever in volunteers ten years after a dose-response study - A complementary study.

A single dose of standard yellow fever (YF) vaccine is considered to provide life-long protection. In this study, we evaluate the seropositivity conferred by lower doses 10 years post-vaccination. In 2009, Bio-Manguinhos/Fiocruz performed a dose-response study with the 17DD yellow fever vaccine, administering the vaccine in the usual mean dose of 27.476 IU and in decreasing doses (10.447 IU, 3.013 IU, 587 IU, 158 IU and 31 IU), with the usual volume and route (0,5 ml subcutaneous). The decreasing doses were obtained by dilution in the laboratory of the manufacturer and the lots in test had standard quality control and were produced by good manufacturing practices (GMP). Around 30 days after the vaccination, doses down to 587 IU had similar immunogenicity and the 158 IU and 31 IU were inferior to the full dose. The seropositivity was maintained for 10 months, except on the 31 IU group. Eight years after, 85 % of 318 participants evaluated in a follow-up, maintained seropositivity that was similar across groups. Consistently, antibody titers in the reduced-dose groups were also comparable to those of the full-dose group. The current study, 10 years later, showed similarity between the vaccine groups (six arms who received the YF vaccine in decreasing doses: 27.476 IU, 10.447 IU, 3.013 IU, 587 IU, 158 IU, 31 IU) both in relation of seropositivity and in the evaluation of the geometric mean titers. The seropositivity rates across subgroups were 83,1%, 90 %, 87 %, 93 %, 83,8% and 85 %, correspondingly. These findings provides further support to the long-term immunogenicity of lower doses. Clinical trial registry: NCT04416477.

Impact of HIV-Related Immune Impairment of Yellow Fever Vaccine Immunogenicity in People Living with HIV-ANRS 12403.

The yellow fever (YF) vaccine is one of the safest and most effective vaccines currently available. Still, its administration in people living with HIV (PLWH) is limited due to safety concerns and a lack of consensus regarding decreased immunogenicity and long-lasting protection for this population. The mechanisms associated with impaired YF vaccine immunogenicity in PLWH are not fully understood, but the general immune deregulation during HIV infection may play an important role. To assess if HIV infection impacts YF vaccine immunogenicity and if markers of immune deregulation could predict lower immunogenicity, we evaluated the association of YF neutralization antibody (NAb) titers with the pre-vaccination frequency of activated and exhausted T cells, levels of pro-inflammatory cytokines, and frequency of T cells, B cells, and monocyte subsets in PLWH and HIV-negative controls. We observed impaired YF vaccine immunogenicity in PLWH with lower titers of YF-NAbs 30 days after vaccination, mainly in individuals with CD4 count <350 cells/mm3. At the baseline, those individuals were characterized by having a higher frequency of activated and exhausted T cells and tissue-like memory B cells. Elevated levels of those markers were also observed in individuals with CD4 count between 500 and 350 cells/mm3. We observed a negative correlation between the pre-vaccination level of CD8+ T cell exhaustion and CD4+ T cell activation with YF-NAb titers at D365 and the pre-vaccination level of IP-10 with YF-NAb titers at D30 and D365. Our results emphasize the impact of immune activation, exhaustion, and inflammation in YF vaccine immunogenicity in PLWH.

Bloodmeals fuel dengue virus replication in the female mosquito Aedes aegypti.

Vector competence defines the ability of a vector to acquire, host, and transmit a pathogen. Understanding the molecular determinants of the mosquitos' competence to host dengue virus (DENV) holds promise to prevent its transmission. To this end, we employed RNA-seq to profile mRNA transcripts of the female Aedes aegypti mosquitos feeding on naïve vs viremic mouse. While most transcripts (12,634) did not change their abundances, 360 transcripts showed decreases. Biological pathway analysis revealed representatives of the decreased transcripts involved in the wnt signaling pathway and hippo signaling pathway. One thousand three hundred fourteen transcripts showed increases in abundance and participate in 21 biological pathways including amino acid metabolism, carbon metabolism, fatty acid metabolism, and oxidative phosphorylation. Inhibition of oxidative phosphorylation with antimycin A reduced oxidative phosphorylation activity and ATP concentration associated with reduced DENV replication in the Aedes aegypti cells. Antimycin A did not affect the amounts of the non-structural proteins 3 and 5, two major components of the replication complex. Ribavirin, an agent that reduces GTP concentration, recapitulated the effects of reduced ATP concentration on DENV replication. Knocking down one of the oxidative phosphorylation components, ATP synthase subunit ß, reduced DENV replication in the mosquitos. In summary, our results suggest that DENV enhances metabolic pathways in the female Aedes aegypti mosquitos to supply nutrients and energy for virus replication. ATP synthase subunit ß knockdown might be exploited to reduce the mosquitos' competence to host and transmit DENV. IMPORTANCE: Through evolution, the mosquito-borne viruses have adapted to the blood-feeding behaviors of their opportunist hosts to fulfill a complete lifecycle in humans and mosquitos. Disruption in the mosquitos' ability to host these viruses offers strategies to prevent diseases caused by them. With the advent of genomic tools, we discovered that dengue virus (DENV) benefited from the female mosquitos' bloodmeals for metabolic and energetic supplies for replication. Chemical or genetic disruption in these supplies reduced DENV replication in the female mosquitos. Our discovery can be exploited to produce genetically modified mosquitos, in which DENV infection leads to disruption in the supplies and thereby reduces replication and transmission. Our discovery might be extrapolated to prevent mosquito-borne virus transmission and the diseases they cause.

Enhanced production of yellow fever virus through tailored culture media optimization.

In the present study, an initial screening was conducted using 12 types of cell culture media, and four media with the best performance were selected for further study. The optimization of four media blend for YFV production was evaluated using an Augmented simplex centroid mixture design. Among all the different models that were investigated, the quadratic model was found to be the most appropriate model for exploring mixture design. It was found that M10 exhibited the greatest impact on YFV production, followed by M9, M4, and M1. The utilization of M1 and M4 media individually yielded higher compared to their blends with other media. The YFV titers were reduced when M1 media was combined with other media. The utilization of M9 and M10 media in combination resulted a higher viral yield compared to their respective concentrations. The optimal ratio for achieving a higher titer of YFV from primary CEFs was found to be approximately 38:62, with M9 and M10 being the most favorable media blend. The use of a media mixture led to a significant increase of virus titer up to 2.6 × 108 PFU/ml or 2 log titer yield, which is equivalent to 1.92 × 105 doses, without any changes to growth conditions or other process factors. This study concluded that the utilization of a mixture design could be efficiently employed to choose the optimal combination of media blends for enhanced viral production from cell culture.