Joakim Esbjörnsson

Background: Human immunodeficiency virus type 1 (HIV-1) is one of the fastest-evolving human pathogens. Understanding HIV-1 transmission, within-host adaptation, and evolutionary dynamics is pivotal for development of interventions and vaccines. HIV-1 infection is generally caused by a single transmitted founder virus (TFV), and TFV sequences are typically obtained using single genome amplification (SGA). However, suboptimal sample quality can cause sequencing failures, representing considerable losses considering the scarcity of acute HIV-1 infection (AHI) samples. Sequencing failures may be mitigated by molecular cloning (MC), which can be less vulnerable to sample quality but more susceptible to polymerase chain reaction (PCR) errors. Here, we explore the feasibility of supplementing SGA with MC data using samples from clinical and research cohorts to determine whether sequence diversity and evolutionary rate estimates are comparable between the techniques. Methods: Plasma samples were selected from participants with documented AHI from an East African research cohort (the International AIDS Vaccine Initiative, 2006–2011) and a clinical cohort from Sweden (1983–2011). SGA and MC sequencing were done on the HIV-1 env V1-V3 region (~940 base pairs). Within-host sequence diversity was determined from maximum likelihood phylogenetic trees, and evolutionary rate by Bayesian phylogenetic analysis. Highlighter plots, Hamming distances, and assessment of star phylogenies were used to quantify TFVs. Results: One hundred participants (median age 30.3 years, 15% female), contributing 350 samples from four longitudinal time points, 10–540 days post-infection, met the inclusion criteria. SGA succeeded on 90% of research cohort and 48% of clinical cohort samples. Comparative analysis of linked SGA and MC data from 10 samples indicated that approximately eight sequences were necessary for diversity estimates. Consistently higher sequence diversity was observed among MC relative to SGA sequences (median [IQR]: 0.009 [0.003, 0.015] and 0.004 [0.001, 0.012] substitutions/site, P = .002), whereas evolutionary rates were comparable between the two methods (0.016 [0.012, 0.019] and 0.011 [0.008, 0.020] substitutions/site/year, P = .232). Five participants with samples obtained within 45 days post-infection were eligible for TFV quantification, and all found to have one TFV using both techniques. Conclusion: MC data is a suitable supplement for SGA-based HIV-1 studies to preserve the value of precious samples for analysis of evolutionary rate, but not for sequence diversity.

A better understanding of the immune responses associated with future viral control in humans during acute HIV-1 infection (AHI) is critical to inform vaccines and immune-based therapeutics. Natural killer (NK) cells and CD8 ⁺ T-cells are pivotal in antiviral defence, yet the dynamics and complementary roles of these effector subsets during AHI with different HIV-1 subtypes remain poorly understood. Access to a unique patient cohort recruited during and post-peak HIV-1 viral load with different HIV-1 subtypes and followed up longitudinally in the absence of antiretroviral therapy up to six years post estimated date of infection (EDI) provided a rare opportunity to fill this knowledge gap. Our data show an early expansion of FcεRγ ⁻ CD57 ⁺ NK cells with classical adaptive traits concomitant with an enhanced capacity for antibody-dependent cellular cytotoxicity (ADCC) and reactivity against HIV-1 antigens. This distinctive NK cell profile was more abundant in donors with subtype A infection compared to non-subtype A, partially driven by elevated pro-inflammatory cytokine levels and changes in the epigenetic landscape. The accumulation of adaptive NK cells during the first month of infection contributed to the optimal activation of CD8 ⁺ T-cells, promoting virus-specific responses. Notably, individuals with higher levels of FcεRγ ⁻ CD57 ⁺ adaptive NK cells during the first month of infection were more likely to exhibit long-term viral control in the absence of ART. These findings underscore the critical role of early, high-magnitude adaptive NK cell responses in CD8 ⁺ T-cell activation and subsequent immune control. This work provides novel insights into the correlates of protective immunity against HIV-1 infection, with implications for preventative or therapeutic vaccine strategies aimed at promoting adaptive NK cell responses. One Sentence Summary: Early expansion of adaptive NK cells during acute HIV-1 infection promotes long-term viral control.

Background: Human immunodeficiency virus type 1 (HIV-1) is one of the fastest evolving human pathogens. Understanding transmission, within-host adaptation, and evolutionary dynamics are pivotal for development of interventions and vaccines. HIV-1 infection is generally caused by one single transmitted founder virus (TFV), and TFV sequences have typically been obtained using single genome amplification (SGA). However, suboptimal sample quality can result in sequencing failures, representing non-trivial losses considering the scarcity of acute HIV-1 infection (AHI) samples. Sequencing failures may be mitigated by molecular cloning (MC), a method that can be less vulnerable to sample quality but more susceptible to PCR errors. Here, we explore the feasibility of supplementing SGA with MC data using samples from clinical and research cohorts to determine whether sequence diversity and evolutionary rate estimates are comparable between the two techniques. Methods: Participants were enrolled in an East African research cohort from the International AIDS Vaccine Initiative 2006-2011 or a clinical cohort from Sweden (1983-2011). SGA and MC sequencing were done on the HIV-1 env V1-V3 region (approximately 940 base pairs). Within-host sequence diversity was determined from maximum likelihood phylogenetic trees and evolutionary rate by Bayesian phylogenetic analysis. Highlighter and Poisson-Fitter tools, Hamming distances, and assessment of star phylogenies were used to quantify TFVs. Results: Participants with AHI (N=100, median age 30.3 years, 15% female) were included, contributing 350 samples from four longitudinal time points 10-540 days post infection. SGA succeeded on 90% of research cohort and 48% of clinical cohort samples. Comparative analysis of linked SGA and MC data from 10 samples indicated that approximately eight sequences were necessary for diversity estimates. Consistently higher sequence diversity was observed among MC relative to SGA sequences (mean±SD 0.009±0.007 and 0.006±0.006 substitutions/site, p<0.001), whereas evolutionary rates were similar between the two methods (mean±SD 0.014±0.006 vs. 0.014±0.009 substitutions/site/year, p=0.673). Five participants with visits within 45 days post infection were eligible for TFV quantification and all found to have one TFV using both MC and SGA data. Conclusion: MC data is a suitable supplement for SGA-based studies to preserve the value of precious samples for evolutionary rate but not sequence diversity analysis.

SARS-CoV-2 can cause clinical and inapparent disease and mortality in several animals cohabitating with humans, and sheep are susceptible to SARS-CoV-2 due to virus–receptor interactions similar to those in humans. Hence, sheep have the potential to be infected, spread, and develop neutralising antibodies (NAbs) against SARS-CoV-2. The aim of this study was to investigate the prevalence of SARS-CoV-2 NAbs in farm animals after natural exposure to the virus. Serum samples were collected from sheep in the Serra da Estrela region in Portugal, both prior to and during the COVID-19 pandemic. The sera were tested by established SARS-CoV-2 pseudovirus systems for multiple SARS-CoV-2 variants (early—Wuhan, mid—Delta, Omicron—BA.1, and late—Omicron XBB, BQ.1.1). Partial neutralisation activity in Pre-pandemic and Mid-pandemic samples was observed, while no NAb activity was observed in Late-pandemic samples tested. Different levels of NAbs were observed between Pre-pandemic samples and those collected during the Mid-pandemic and Late-pandemic periods (p ≤ 0.01). Our results indicate that SARS-CoV-2 cross-species transmission may have occurred through human–sheep contacts on sheep farms during the pandemic, and that farm animals could contribute to the One Health Approach in zoonotic virus surveillance and pandemic preparedness.

The complex dynamics of protein expression in plasma during hyperacute HIV-1 infection and its relation to acute retroviral syndrome, viral control, and disease progression are largely unknown. Here, we quantify 1293 blood plasma proteins from 157 longitudinally linked plasma samples collected before, during, and after hyperacute HIV-1 infection of 54 participants from four sub-Saharan African countries. Six distinct longitudinal expression profiles are identified, of which four demonstrate a consistent decrease in protein levels following HIV-1 infection. Proteins involved in inflammatory responses, immune regulation, and cell motility are significantly altered during the transition from pre-infection to one month post-infection. Specifically, decreased ZYX and SCGB1A1 levels, and increased LILRA3 levels are associated with increased risk of acute retroviral syndrome; increased NAPA and RAN levels, and decreased ITIH4 levels with viral control; and increased HPN, PRKCB, and ITGB3 levels with increased risk of disease progression. Overall, this study provides insight into early host responses in hyperacute HIV-1 infection, and present potential biomarkers and mechanisms linked to HIV-1 disease progression and viral load.

Objective: To estimate the prevalence of the curable sexually transmitted infections (STIs) Chlamydia trachomatis , Neisseria gonorrhoeae , Mycoplasma genitalium , Trichomonas vaginalis and Treponema pallidum , to identify associated risk factors and to assess ciprofloxacin resistance in N. gonorrhoeae -positive specimens among female sex workers (FSWs) in Guinea-Bissau. Methods: For this cross-sectional study, FSWs were recruited from October 2014 to May 2019. A questionnaire on STI risk factors was completed by the study participants, and the women were asked to provide a vaginal swab for nucleic acid amplification tests for C. trachomatis , N. gonorrhoeae , M. genitalium , T. vaginalis (Aptima, Hologica), as well as a blood sample for T. pallidum serological testing and discriminatory HIV-testing. The prevalence of STIs was determined, and multivariate logistic regression was used to identify STI risk factors. Results: The study included 467 women. The prevalence of current infection with any curable STI was 46.7%, and the most common pathogen was T. vaginalis (26.3%), followed by M. genitalium (21.9%), C. trachomatis (11.8%), N. gonorrhoeae (10.1%) and T. pallidum (2.8%). The proportion of asymptomatic infections among the diagnosed STIs was 61.8%, 61.5%, 55.3%, 55.3% and 52.2% for C. trachomatis, T. pallidum, N. gonorrhoeae, T. vaginalis and M. genitalium, respectively . The prevalence of the gyrA S91F mutation conferring ciprofloxacin resistance in N. gonorrhoeae -positive specimens was 84.0%. Significant risk factors for having a curable STI were age and HIV-1 infection, while use of female condoms was a protective factor. Conclusion: This study demonstrated that the prevalence of curable STIs was high among FSWs in Guinea-Bissau during the study period, indicating an unmet need for STI services. Moreover, the results indicated that symptomatic treatment might be insufficient, highlighting a need for periodic aetiological testing to facilitate detection of asymptomatic as well as symptomatic STIs to stop ongoing transmission.

The asymptomatic disease stage in HIV-2 infection is approximately twice as long compared to in HIV-1 infection, still the majority of HIV-2 infected individuals progress to AIDS in the absence of antiretroviral treatment. In this study, we applied data-independent acquisition mass spectrometry analysis of blood plasma samples collected from HIV negative, and HIV-1 or HIV-2 infected individuals in Guinea-Bissau with an estimated date of HIV infection, to explore associations between plasma proteome alterations and HIV disease progression. In total, 609 proteins were quantified and mapped towards publicly available data on tissue-enhanced genes, to provide insight on the tissue-specific origin of the detected proteins. Here we identified ten proteins that could differentiate between faster and slower HIV disease progression. The analysis also suggested a larger leakage of proteins from the sigmoid colon in HIV-1 compared to HIV-2 infection. Moreover, the levels of sigmoid colon and spleen tissue proteins were associated with disease progression among all HIV infected individuals. In conclusion, these results encourage further research on the role of both target and bystander cells in HIV disease progression.

Escape from cytotoxic T lymphocyte (CTL) responses toward HIV-1 Gag and Nef has been associated with reduced control of HIV-1 replication in adults. However, less is known about CTL-driven immune selection in infants as longitudinal studies of infants are limited. Here, 1,210 gag and 1,264 nef sequences longitudinally collected within 15 months after birth from 14 HIV-1 perinatally infected infants and their mothers were analyzed. The number of transmitted founder (T/F) viruses and associations between virus evolution, selection, CTL escape, and disease progression were determined. The analyses indicated that a paraphyletic-monophyletic relationship between the mother-infant sequences was common (80%), and that the HIV-1 infection was established by a single T/F virus in 10 of the 12 analyzed infants (83%). Furthermore, most HIV-1 CTL escape mutations among infants were transmitted from the mothers and did not revert during the first year of infection. Still, immune-driven selection was observed at approximately 3 months after HIV-1 infection in infants. Moreover, virus populations with CTL escape mutations in gag evolved faster than those without, independently of disease progression rate. These findings expand the current knowledge of HIV-1 transmission, evolution, and CTL escape in infant HIV-1 infection and are relevant for the development of immune-directed interventions in infants. IMPORTANCE: Despite increased coverage in antiretroviral therapy for the prevention of perinatal transmission, paediatric HIV-1 infection remains a significant public health concern, especially in areas of high HIV-1 prevalence. Understanding HIV-1 transmission and the subsequent virus adaptation from the mother to the infant’s host environment, as well as the viral factors that affect disease outcome, is important for the development of early immune-directed interventions for infants. This study advances our understanding of vertical HIV-1 transmission, and how infant immune selection pressure is shaping the intra-host evolutionary dynamics of HIV-1.

HIV-1 remains incurable and there is no effective vaccine towards the infection. A main challenge for this is the lack of a holistic understanding of the myriad of complex virus-host interactions during hyperacute HIV-1 infection (hAHI), and how these contribute to tissue damage and pathogenesis. Here, 1293 blood plasma proteins were quantified from 157 linked plasma samples collected before, during, and after hAHI of 54 volunteers from four sub-Saharan African countries. Six distinct longitudinal expression profiles were identified, of which four demonstrated a consistent decrease in protein levels following HIV-1 infection. Differentially expressed proteins were involved in inflammation, innate immunity, cell motility, and actin cytoskeleton reorganisation. Specifically, decreased levels of Zyxin, Secretoglobin family 1A member 1, and Pro-platelet basic protein were associated with acute retroviral syndrome; Rho GTPase activating protein 18, Annexin A1, and Lipopolysaccharide binding protein with viral load; and Hepsin, Protein kinase C beta, and Integrin subunit beta 3 with disease progression. This is the first holistic characterisation of within-patient blood plasma proteome dynamics during the first weeks of HIV-1 infection and presents multiple potential blood biomarkers and targets for prophylactic and therapeutic HIV-1 interventions.

The role of human leukocyte antigen (HLA) class I and killer immunoglobulin-like receptor molecules in mediating acute retroviral syndrome (ARS) during human immunodeficiency virus type 1 (HIV-1) infection is unclear. Among 72 sub-Saharan African adults, HLA-A*23 was associated with lower odds of ARS (adjusted odds ratio, 0.10 [95% confidence interval, .01–.48]; P = .009), which warrants further studies to explore its role on HIV-1–specific immunopathogenesis.