Amy L. Ellis-Connell

The emergency department (ED) is a common site for respiratory viral surveillance, but air sampling has not been thoroughly explored in this setting. In this study, we observed a significant association between COVID-19, influenza A, and RSV detection via air sampling in an ED waiting room and clinical viral testing.

Advances in air-based surveillance of pathogen genetic material are hindered by reliance on centralized, time-consuming molecular techniques. Point-of-care (POC) diagnostic platforms, like the Cepheid® GeneXpert®, offer rapid, simplified testing in clinical settings but have not been evaluated for use with air samples. Here, we paired the ThermoFisher™ AerosolSense™ air sampler with the Xpert® Xpress SARS-CoV-2/Flu/RSV Plus test to evaluate near-real-time air surveillance. To assess analytical sensitivity, we spiked collection substrates with inactivated viruses and performed overnight sampling using the air sampler. As few as 10 copies of influenza A/B (IAV/IBV) and RSV applied to the substrate were detectable by GeneXpert, while SARS-CoV-2 required at least 100 copies for detection. Longitudinal air surveillance was conducted across congregate settings in Columbia, Missouri, and Madison, Wisconsin, in 2024-2025, collecting 281 air samples. SARS-CoV-2 was detected most frequently, followed by IAV. To assess concordance, 191 samples with paired GeneXpert and RT-qPCR results were analyzed across multiple Ct value cutoffs. Agreement between GeneXpert and RT-qPCR for SARS-CoV-2 was fair to moderate (κ = 0.306–0.443). Optimal GeneXpert Ct cutoffs for the best balance between sensitivity and specificity, determined using analyses such as Youden’s index, were site-specific: 45 for Wisconsin (67% sensitivity, 83% specificity) and 41 for Missouri (76% sensitivity, 62% specificity), reflecting differences in laboratory protocols. For IAV, agreement was moderate (κ = 0.56) with GeneXpert Ct cutoff of 40, achieving 85% sensitivity and 81% specificity. Further studies across diverse settings and viral targets are needed to establish GeneXpert’s role in routine air surveillance. Importance: This study demonstrates that point-of-care molecular testing with the Cepheid® GeneXpert® provides a faster alternative to the time-consuming molecular workflows currently used for detecting airborne respiratory pathogens. The GeneXpert provides results within 37 minutes after sample addition to the Xpress CoV-2/Flu/RSV test cartridge (following approximately 20 minutes for sample elution), compared to the multiple hours or days required by laboratory-based methods, enabling mitigation measures before widespread transmission occurs. Integrating GeneXpert testing with air sampling offers a simple, semi-automated approach for pathogen detection in healthcare and community settings. Because GeneXpert instruments are already deployed in over 180 countries, including clinics in low- and middle-income settings, this approach can extend air sampling–based surveillance to regions where individual patient testing is not feasible. These workflows can also be adapted to detect other pathogens with point-of-care assays.

Air sampling is a non-invasive alternative to individual testing for respiratory pathogens. Alternative methods to the “gold standard” quantitative reverse transcription-PCR (qRT-PCR) are required to enable higher throughput, lower cost, and more multiplexed detection of pathogens. The multiplexed CRISPR-Cas13 CARMEN Respiratory Viral Panel (RVP) was described previously for high-throughput detection of nine respiratory pathogens from nasal swab samples. Here, we modified and optimized the CARMEN RVP assay to overcome the unique challenges of air samples, including low biomass and environmental inhibitors. We monitored for SARS-CoV-2 and influenza A (Flu A) via qRT-PCR in air samples from 15 schools within Dane County, Wisconsin (USA), during the 2023–2024 school year. SARS-CoV-2 was detectable throughout the entire sampling period, while Flu A detection was seasonal from November 2023 to March 2024. We then analyzed a subset of samples from seven schools using an optimized CARMEN RVP assay for air surveillance (RVP_air) and compared the results to qRT-PCR. The RVP_air assay detected several additional pathogens beyond our primary targets. The frequencies and patterns of SARS-CoV-2 positivity, but not Flu A positivity, were similar between qRT-PCR and RVP_air across the 2023–2024 sampling period. We developed a secondary panel (RVP_air_flu) to better detect both H1N1 and H3N2 subtypes. Finally, we compared air sample results to clinical nasal swabs collected from the same school district. For several pathogens (SARS-CoV-2, HCoV-OC43, Flu A), positive air detections coincided with positive nasal swabs. These findings demonstrate that the RVP_air assay can effectively detect airborne pathogens from infected individuals within indoor spaces. IMPORTANCE: Air sampling offers a cost-effective alternative to individual testing for respiratory pathogens within congregate settings. Optimization and use of multi-pathogen assays are especially valuable for capturing the breadth of pathogens that may be present simultaneously in the same space. The modified CARMEN RVP assays (RVP_air and RVP_air_flu) detected SARS-CoV-2 and Flu A during similar sampling time periods compared to qRT-PCR, while also detecting several additional respiratory pathogens (seasonal coronaviruses, respiratory syncytial virus). Importantly, pathogens detected from air samples corresponded to those detected from nasal swabs collected from individuals in the same spaces. Together, these findings highlight the utility of the RVP_air and RVP_air_flu assays as alternatives to qRT-PCR for environmental surveillance, with applications extending to other congregate spaces (hospitals, long-term care facilities) and high-risk settings, better informing communities and improving public health.

Air sampling is a non-invasive alternative to individual testing for respiratory pathogens. Alternative methods to the “gold standard” quantitative RT-PCR (qRT-PCR) are required to enable higher throughput, lower cost, and more multiplexed detection of pathogens. The multiplexed CRISPR-Cas13 CARMEN Respiratory Viral Panel (RVP) was described previously for high-throughput detection of nine respiratory pathogens from nasal swab samples. Here, we modified and optimized the CARMEN RVP assay to overcome the unique challenges of air samples, including low biomass and environmental inhibitors. We monitored for SARS-CoV-2 and influenza A (Flu A) via qRT-PCR in air samples from 15 schools within Dane County, Wisconsin (USA) during the 2023-2024 school year. SARS-CoV-2 was detectable throughout the entire sampling period, while Flu A detection was seasonal from November 2023 to March 2024. We then analyzed a subset of samples from seven schools using an optimized CARMEN RVP assay for air surveillance (RVP_air) and compared results to qRT-PCR. The RVP_air assay detected several additional pathogens beyond our primary targets. The frequencies and patterns of SARS-CoV-2 positivity, but not Flu A, were similar between qRT-PCR and RVP_air across the 2023-2024 sampling period. We developed a secondary panel (RVP_air_flu) to better detect both H1N1 and H3N2 subtypes. Finally, we compared air sample results to clinical nasal swabs collected from the same school district. For several pathogens (SARS-CoV-2, HCoV-OC43, Flu A), positive air detections coincided with positive nasal swabs. These findings demonstrate that the RVP_air assay can effectively detect airborne pathogens from infected individuals within indoor spaces. IMPORTANCE Air sampling offers a cost-effective alternative to individual testing for respiratory pathogens within congregate settings. Optimization and use of multi-pathogen assays are especially valuable for capturing the breadth of pathogens that may be present simultaneously in the same space. The modified CARMEN RVP assays (RVP_air and RVP_air_flu) detected SARS-CoV-2 and Flu A during similar sampling time periods compared to qRT-PCR, while also detecting several additional respiratory pathogens (seasonal Coronaviruses, Respiratory Syncytial Virus). Importantly, pathogens detected from air samples corresponded to those detected from nasal swabs collected from individuals in the same spaces. Together, these findings highlight the utility of the RVP_air and RVP_air_flu assays as alternatives to qRT-PCR for environmental surveillance, with applications extending to other congregate spaces (hospitals, long-term care facilities) and high-risk settings, better informing communities and improving public health.

Sustainable HIV remission after antiretroviral therapy (ART) withdrawal, or post-treatment control (PTC), remains a top priority for HIV treatment. We observed surprising PTC in an MHC-haplomatched cohort of MHC-M3+ SIVmac239+ Mauritian cynomolgus macaques (MCMs) initiated on ART at two weeks post-infection (wpi). For six months after ART withdrawal, we observed undetectable or transient viremia in seven of eight MCMs. In vivo depletion of CD8α+ cells induced rebound in all animals, indicating the PTC was mediated, at least in part, by CD8α+ cells. We found that MCMs had smaller acute viral reservoirs than a cohort of identically infected rhesus macaques, a population that rarely develops PTC. The mechanisms by which unusually small viral reservoirs and CD8α+ cell-mediated virus suppression enable PTC can be investigated using this MHC-haplomatched MCM model. Further, defining the immunologic mechanisms that engender PTC in this model may identify therapeutic targets for inducing durable HIV remission in humans.

Vaccine strategies aimed at eliciting HIV-specific CD8+ T cells are one major target of interest in HIV functional cure strategies. We hypothesized that CD8+ T cells elicited by therapeutic vaccination during antiretroviral therapy (ART) would be recalled and boosted by treatment with the IL-15 superagonist N-803 after ART discontinuation. We intravenously immunized four SIV+ Mauritian cynomolgus macaques (MCM) receiving ART with vesicular stomatitis virus (VSV), modified vaccinia virus Ankara strain (MVA), and recombinant adenovirus serotype 5 (rAd-5) vectors all expressing SIVmac239 Gag. Immediately after ART cessation, these animals received three doses of N-803. Four control animals received no vaccines or N-803. The vaccine regimen generated a high magnitude of Gag-specific CD8+ T cells that were proliferative and biased toward an effector memory phenotype. We then compared cells elicited by vaccination (Gag-specific) to cells elicited by SIV infection and unaffected by vaccination (Nef-specific). We found that N-803 treatment enhanced both the frequencies of bulk and proliferating antigen-specific CD8+ T cells elicited by vaccination and the antigen-specific CD8+ T cells elicited by SIV infection. In sum, we demonstrate that a therapeutic heterologous prime-boost-boost (HPBB) vaccine can elicit antigen-specific effector memory CD8+ T cells that are boosted by N-803.

The IL-15 superagonist N-803 has been shown to enhance the function of CD8 T cells and NK cells. We previously found that in a subset of vaccinated, ART-naïve, SIV+ rhesus macaques, N-803 treatment led to a rapid but transient decline in plasma viremia that positively correlated with an increase in the frequency of CD8 T cells. Here we tested the hypothesis that prophylactic vaccination was required for N-803 mediated suppression of SIV plasma viremia. We vaccinated rhesus macaques with a DNA prime/Ad5 boost regimen using vectors expressing SIVmac239 gag, with or without a plasmid expressing IL-12, or left them unvaccinated. Animals were then intravenously infected with SIVmac239M. Six months after infection, animals were treated with N-803. We found no differences in control of plasma viremia during N-803 treatment between vaccinated and unvaccinated macaques. Furthermore, the SIV-specific CD8 T cells displayed no differences in frequency or ability to traffic to the lymph nodes. Interestingly, when we divided the SIV+ animals based on plasma viral load set-point prior to N-803 treatment, N-803 increased the frequency of SIV-specific T cells expressing ki-67+ and granzyme B+ in animals with low plasma viremia (10 ⁴ copies/mL;SIV non-controllers). In addition, Gag-specific CD8 T cells from the SIV+ controllers had a greater increase in CD107a+CD8+ T cells when compared to SIV+ non-controllers. Overall, our results indicate that N-803 is most effective in SIV+ animals with a pre-existing immunological ability to control SIV replication.

Immunomodulatory drugs could contribute to a functional cure for Human Immunodeficiency Virus (HIV). Interleukin-15 (IL-15) promotes expansion and activation of CD8⁺ T cell and natural killer (NK) cell populations. In one study, an IL-15 superagonist, N-803, suppressed Simian Immunodeficiency Virus (SIV) in non-human primates (NHPs) who had received prior SIV vaccination. However, viral suppression attenuated with continued N-803 treatment, partially returning after long treatment interruption. While there is evidence of concurrent drug tolerance, immune regulation, and viral escape, the relative contributions of these mechanisms to the observed viral dynamics have not been quantified. Here, we utilize mathematical models of N-803 treatment in SIV-infected macaques to estimate contributions of these three key mechanisms to treatment outcomes: 1) drug tolerance, 2) immune regulation, and 3) viral escape. We calibrated our model to viral and lymphocyte responses from the above-mentioned NHP study. Our models track CD8⁺ T cell and NK cell populations with N-803-dependent proliferation and activation, as well as viral dynamics in response to these immune cell populations. We compared mathematical models with different combinations of the three key mechanisms based on Akaike Information Criterion and important qualitative features of the NHP data. Two minimal models were capable of reproducing the observed SIV response to N-803. In both models, immune regulation strongly reduced cytotoxic cell activation to enable viral rebound. Either long-term drug tolerance or viral escape (or some combination thereof) could account for changes to viral dynamics across long breaks in N-803 treatment. Theoretical explorations with the models showed that less-frequent N-803 dosing and concurrent immune regulation blockade (e.g. PD-L1 inhibition) may improve N-803 efficacy. However, N-803 may need to be combined with other immune therapies to countermand viral escape from the CD8⁺ T cell response. Our mechanistic model will inform such therapy design and guide future studies.

Tuberculosis (TB) is the leading infectious cause of death among people living with HIV. People living with HIV are more susceptible to contracting Mycobacterium tuberculosis and often have worsened TB disease. Understanding the immunologic defects caused by HIV and the consequences it has on M. tuberculosis coinfection is critical in combating this global health epidemic. We previously showed in a model of SIV and M. tuberculosis coinfection in Mauritian cynomolgus macaques (MCM) that SIV/M. tuberculosis–coinfected MCM had rapidly progressive TB. We hypothesized that pre-existing SIV infection impairs early T cell responses to M. tuberculosis infection. We infected MCM with SIVmac239, followed by coinfection with M. tuberculosis Erdman 6 mo later. Although similar, TB progression was observed in both SIV+ and SIV-naive animals at 6 wk post–M. tuberculosis infection; longitudinal sampling of the blood (PBMC) and airways (bronchoalveolar lavage) revealed a significant reduction in circulating CD4+ T cells and an influx of CD8+ T cells in airways of SIV+ animals. At sites of M. tuberculosis infection (i.e., granulomas), SIV/M. tuberculosis–coinfected animals had a higher proportion of CD4+ and CD8+ T cells expressing PD-1 and TIGIT. In addition, there were fewer TNF-producing CD4+ T cells in granulomas of SIV/M. tuberculosis–coinfected animals. Taken together, we show that concurrent SIV infection alters T cell phenotypes in granulomas during the early stages of TB disease. As it is critical to establish control of M. tuberculosis replication soon postinfection, these phenotypic changes may distinguish the immune dysfunction that arises from pre-existing SIV infection, which promotes TB progression.

There were an estimated 10 million new cases of tuberculosis (TB) disease in 2019. While over 90% of individuals successfully control Mycobacterium tuberculosis (Mtb) infection, which causes TB disease, HIV co-infection often leads to active TB disease. Despite the co-endemic nature of HIV and TB, knowledge of the immune mechanisms contributing to the loss of control of Mtb replication during HIV infection is lacking. Mucosal-associated invariant T (MAIT) cells are innate-like T cells that target and destroy bacterially-infected cells and may contribute to the control of Mtb infection. Studies examining MAIT cells in human Mtb infection are commonly performed using peripheral blood samples. However, because Mtb infection occurs primarily in lung tissue and lung-associated lymph nodes, these studies may not be fully translatable to the tissues. Additionally, studies longitudinally examining MAIT cell dynamics during HIV/Mtb co-infection are rare, and lung and lymph node tissue samples from HIV+ patients are typically unavailable. Nonhuman primates (NHP) provide a model system to characterize MAIT cell activity during Mtb infection, both in Simian Immunodeficiency Virus (SIV)-infected and SIV-naïve animals. Using NHPs allows for a more comprehensive understanding of tissue-based MAIT cell dynamics during infection with both pathogens. NHP SIV and Mtb infection is similar to human HIV and Mtb infection, and MAIT cells are phenotypically similar in humans and NHPs. Here, we discuss current knowledge surrounding MAIT cells in SIV and Mtb infection, how SIV infection impairs MAIT cell function during Mtb co-infection, and knowledge gaps to address.