Respiratory syncytial virus (RSV) remains a major cause of severe respiratory disease worldwide, particularly affecting young children and immunocompromised individuals, highlighting the need for additional therapeutic strategies. In this study, the antiviral activity of the Petasites hybridus extract Ze 339 was investigated in RSV-infected cell-culture models. Antiviral efficacy was assessed using plaque reduction assays, reporter virus analyses, and proteomic profiling to elucidate potential mechanisms of action. Ze 339 potently reduced the infectivity of both RSVA and RSVB in vitro and retained antiviral activity when administered up to six hours post-infection, resulting in markedly reduced plaque formation and viral protein biosynthesis without inducing cytotoxicity. Proteomic analyses revealed that Ze 339 modulates host cell pathways associated with reduced cell proliferation, attenuated immune signaling, and enhanced cholesterol and lipid metabolism. These changes were more pronounced in infected than in uninfected cells and coincided with a marked downregulation of viral proteins. The observed proteomic signature suggests a host-directed antiviral effect and identifies altered lipid metabolism and cell-cycle-associated pathways as potential contributors to reduced RSV replication. Taken together, these findings demonstrate the antiviral activity of Ze 339 against RSV and support the hypothesis that modulation of host cell pathways contributes to its antiviral effects, providing a rationale for further evaluation of Ze 339 as a repurposed therapeutic candidate for RSV infection.

BACKGROUND:Children and adolescents with HIV have lower treatment success than adults. Suboptimal adherence and resistance to antiretroviral therapy (ART) are known aetiological factors. This preplanned analysis in the GIVE MOVE trial (NCT04233242) describes drug resistance patterns in children and adolescents in Lesotho and Tanzania. MATERIALS AND METHODS:GIVE MOVE randomized children and adolescents (6 months to below 19 years) with recent viraemia whilst taking ART to genotypic resistance testing (GRT)-informed care or usual care. Here, we conducted additional post-hoc GRT on stored samples from both groups and included participants with at least one successful resistance test. We assessed the number of drugs predicted to be active in participants' three-drug ART regimens and resistance-associated mutations. RESULTS:Amongst 137 participants, the majority were female (58%) and lived in Lesotho (77%). At their initial GRT, 69/137 (50%) were receiving protease inhibitor-based, 59/137 (43%) dolutegravir-based and 9/137 (7%) efavirenz-based ART. At that time, 80/137 (58%) participants had three, whilst 8/137 (6%) had two, 36/137 (26%) had one and 13/137 (9%) had no drugs predicted to be active in their regimens. Seventeen (12%) participants had resistance against their ART core agent, including one with high-level dolutegravir resistance.Across 312 detected resistance-associated mutations (222 major, 90 accessory), 146 conferred resistance to non-nucleoside reverse transcriptase inhibitors, 127 to nucleoside reverse transcriptase inhibitors, 28 to protease inhibitors and 11 to integrase strand transfer inhibitors. CONCLUSION:Given that more than half had an ART regimen predicted to be fully active, most viraemia in children and adolescents could not be explained by resistance.Registration: The GIVE MOVE trial was registered on Clinicaltrials.gov NCT04233242.

Although subtype B historically dominated HIV-1 epidemics in western and parts of central Europe, the WHO European region is now characterised by a heterogeneous mixture of HIV-1 variants including non-B subtypes, circulating recombinant forms (CRFs), and unique recombinant forms. This diversification reflects repeated introductions through migration, local onward transmission, and ongoing recombination. Subtype A, particularly sub-subtype A6, continues to dominate much of eastern Europe, where the largest regional burden of HIV-1 is concentrated. Across Europe, marked regional differences persist, with subtype B remaining common in western Europe, diverse non-B variants and CRFs contributing to western and central European epidemics, and recombinant forms increasingly recognised across all regions. Emerging clinical and laboratory data suggest that sub-subtype A6 might have an increased propensity to develop resistance under cabotegravir pressure, with potential implications for long-acting cabotegravir-rilpivirine treatment and cabotegravir-based pre-exposure prophylaxis in regions where A6 circulates widely. Subtype diversity might also affect virulence, reservoir dynamics, and susceptibility to broadly neutralising antibodies and other novel therapeutic approaches. In this Review, we summarise the changing epidemiology of HIV-1 variants in Europe and outline how growing viral diversity should inform prevention, treatment, and surveillance strategies.

Purpose The prospective Viral load Cohort North-East Lesotho (VICONEL) aims to support clinical management and generate scientific evidence to inform HIV care. Specifically, VICONEL allows for the monitoring of HIV treatment outcomes and health system performance, encompasses a biobank for further research with routinely collected blood plasma samples of consenting participants and provides a valuable framework for nested observational and interventional studies.Participants VICONEL captures routine viral load test results alongside associated demographic and treatment information among people in care for HIV in Lesotho, southern Africa. As of December 2023, it encompasses all viral load testing from 24 healthcare facilities in two districts of Lesotho.Findings to date From January 2016 to December 2023, 137 660 viral load test results were available for 29 380 participants. At the time of the last viral load test, median age was 42 years (IQR: 33–53); 18 511 (63%) were adult women, 10 029 (34%) adult men and 835 (3%) children <15 years (age/sex missing for 5) and median time taking antiretroviral therapy (ART) was 6.4 years (IQR 3.2–9.9). Overall, the proportion of cohort participants with viral suppression to <1000 copies/mL has continually exceeded 90% and has been above 95% since 2020; however, this proportion has consistently been lower among children. Sex, age category/ART regimen core agent (combined variable), time since ART initiation and district were independently associated with viraemia.Future plans VICONEL offers potential for (1) further digitalisation and automation of results sharing at the client, facility and district/national level, (2) integration of additional clinical and diagnostic data, including comorbidities and drug resistance and (3) embedding randomised trials.

Vaccines have played a central role in combating the COVID-19 pandemic, but newly emerging SARS-CoV-2 variants are increasingly evading first-generation vaccine protection. To address this challenge, we designed “single-cycle infection SARS-CoV-2 viruses” (SCVs) that lack essential viral genes, possess distinctive immune-modulatory features, and exhibit an excellent safety profile in the Syrian hamster model. Animals intranasally vaccinated with an Envelope-gene-deleted vaccine candidate were fully protected against an autologous challenge with the SARS-CoV-2 virus through systemic and mucosal humoral immune responses. Additionally, the deletion of immune-downregulating viral genes in the vaccine construct prevented challenge virus transmission to contact animals. Moreover, vaccinated animals displayed neither tissue inflammation nor lung damage. Consequently, SCVs hold promising potential to induce potent protection against COVID-19, surpassing the immunity conferred by natural infection, as demonstrated in human immune cells.

BACKGROUND:Children and adolescents with HIV taking antiretroviral therapy (ART) have high rates of viraemia. We assessed if genotypic resistance testing (GRT) to inform onward treatment improved treatment outcomes in Lesotho and Tanzania, two countries with little access to GRT. METHODS:The Genotype-Informed Versus Empirical Management of Viremia (GIVE MOVE) open-label, parallel-group randomised controlled trial enrolled children and adolescents with HIV between the ages of 6 months and 19 years, taking ART, and with a viral load at least 400 copies per mL. Participants were recruited from ten clinical centres and hospitals in Lesotho and Tanzania. Participants were electronically randomly allocated 1:1 to receive either GRT with expert recommendation (GRT group) or repeat viral-load testing and empirical onward treatment (usual care group). Participants and study staff were not masked, but the endpoint committee and laboratory staff conducting viral-load testing were. Participants in both groups received at least three sessions of enhanced adherence counselling, and in the GRT group, blood for GRT assessed via Sanger sequencing was drawn at enrolment. The composite primary endpoint was death, hospitalisation, a new WHO HIV clinical stage 4 event, or not having documented viral suppression of less than 50 copies per mL at 36 weeks in the modified intention-to-treat population, which excluded participants who were retrospectively found to be ineligible after randomisation. Serious adverse events were analysed in the modified intention-to-treat population. The trial was registered with ClinicalTrials.gov (NCT04233242) and the trial status is completed. FINDINGS:Between March 3, 2020, and July 5, 2022, 286 participants were enrolled and 284 were included in the modified intention-to-treat analysis (144 in the GRT group and 140 in the usual care group). Of these participants, 158 (56%) were female and 126 (44%) were male. Five (3%) in the GRT group and four (3%) in the usual care group did not complete follow-up but were included in the primary analysis. The median age across both groups was 14 years (IQR 9-16). The composite primary endpoint occurred in 67 (47%) participants in the GRT group and 73 (52%) in the usual care group (adjusted odds ratio 0·79 [95% CI 0·49 to 1·27]; adjusted risk difference -0·06 [95% CI -0·17 to 0·06]; p=0·34); all participants reaching the composite primary endpoint had no documented viral suppression at 36 weeks. No deaths were recorded, and only one clinical stage 4 event requiring hospitalisation occurred (in the usual care group); this was the only serious adverse event recorded in the study. INTERPRETATION:GRT-informed management did not significantly improve treatment outcomes for children and adolescents with viraemia while taking ART. FUNDING:Fondation Botnar, Swiss National Science Foundation, and Gottfried and Julia Bangerter-Rhyner Foundation. TRANSLATIONS:For the Sesotho and Swahili translations of the abstract see Supplementary Materials section.

Currently, HIV morbidity and mortality in sub-Saharan Africa remain a huge concern and awaiting interventions. Even though the combination antiretroviral therapy (cART) has recorded significant success, drug resistance and limited access to available therapeutics are major factors responsible for the low impact of cART in several African communities. Herein, as part of our continuous effort on the investigation of bioactive metabolites of Ochna rhizomatosa, we report the isolation of a new flavonoid; Rhizomatoflavonoid D (1), alongside with four known ones (2–5). The structures of these compounds were elucidated by using spectroscopic techniques (1H NMR, 13C NMR, HSQC, HMBC, 1H-1H COSY, and ROESY) and mass spectrometry. The antiviral activity of the resulting compounds was assessed using deCIPhR assay run in parallel with the Alamar Blue based cytotoxicity assay. This assay revealed a moderate activity for compound 4 (72

Background Current COVID-19 vaccines primarily target the Spike protein of defined virus variants, offering limited protection against emerging variants in immunocompetent individuals. Similarly, protective immunity following natural SARS-CoV-2 infection is variable and of short duration, raising concerns about immunocompromised individuals' vaccination strategies.Methods This prospective multicenter study examined 66 sera from 59 immunocompromised and 451 sera from 215 immunocompetent individuals from different pandemic periods. We establish and validate a live virus-based neutralization assay to determine the virus-inactivating potential against ancestral and current SARS-CoV-2 isolates.Results Our virus-based neutralization assay demonstrated superior performance over surrogate neutralization assays. We found strong but transient immunity after complete vaccination schemes, with single doses providing minimum neutralization, regardless of vaccine type. Combining vaccination-induced immunity with SARS-CoV-2 infection before or after vaccination yielded higher neutralizing titers than vaccination or infection alone, consistent across both study groups. Additional doses after a full vaccination course restored neutralization levels.Conclusions Potentially protective SARS-CoV-2 neutralization is reliably induced in immunocompromised individuals by prior attenuation of immunosuppression. First-generation vaccines protect against various SARS-CoV-2 variants in immunocompetent individuals, with effective cross-neutralization demonstrated up to the Delta variant but largely absent for later Omicron variants. Continuous vaccine updates are necessary to address emerging SARS-CoV-2 variants. This study evaluated the impact of repeated vaccination and SARS-CoV-2 infection on the virus neutralization capacity in immunocompromised and immunocompetent individuals during different episodes of the pandemic. Neutralization over time was categorized by age, immunization history, and vaccination status.

Even during extended periods of effective immunological control, a substantial dynamic of the viral genome can be observed in different cellular compartments in HIV-1 positive individuals, indicating the persistence of active viral reservoirs. To obtain further insights, we studied changes in the proviral as well as in the viral HIV-1 envelope (Env) sequence along with transcriptional, translational and viral outgrowth activity as indicators for viral dynamics and genomic intactness. Our study identified distinct reservoir patterns that either represented highly sequence-diverse HIV-1 populations or only a single / few persisting virus variants. The single dominating variants were more often found in individuals starting ART during early infection phases, indicating that early treatment might limit reservoir diversification. At the same time, more sequence-diverse HIV reservoirs correlated with a poorer immune status, indicated by lower CD4 count, a higher number of regimen changes and more co-morbidities. Furthermore, we noted that in T-cell populations in the peripheral blood, replication-competent HIV-1 is predominantly present in Lymph node homing TN (naïve) and TCM (central memory) T cells. Provirus genomes archived in TTM (transitional memory) and TEM (effector memory) T cells more frequently tended to carry inactivating mutations and, population-wise, possess changes in the genetic diversity. These discriminating properties of the viral reservoir in T-cell subsets may have important implications for new early therapy strategies, underscoring the critical role of early therapy in preserving robust immune surveillance and constraining the viral reservoir.

Purpose The prospective Viral load Cohort North-East Lesotho (VICONEL) aims to support clinical management and generate scientific evidence to inform HIV care. Specifically, VICONEL allows for monitoring of HIV treatment outcomes and health system performance, encompasses a biobank for further research with routinely collected blood plasma samples of consenting participants, and provides a valuable framework for nested observational and interventional studies. Participants VICONEL captures routine viral load test results alongside associated demographic and treatment information among people in care for HIV in Lesotho, southern Africa. As of December 2022, it encompasses all viral load testing from 23 healthcare facilities in two districts of Lesotho. Findings to date From January 2016 to December 2022, 114’838 viral load test results were available for 27,472 participants. At the time of the last viral load test, median age was 42 years (interquartile range [IQR]: 33-53); 17,324 (63%) were adult women, 9,273 (34%) adult men, and 870 (3%) children <15 years (age/sex missing for 5); and median time taking antiretroviral therapy (ART) was 6.0 years (IQR 3.0-9.2). Overall, the proportion of cohort participants with viral suppression to <1,000 copies/mL has continually exceeded 90% and has been above 95% since 2020; however, this proportion has consistently been lower among children. Sex, age category / ART regimen core agent (combined variable), time since ART initiation, and district were independently associated with viraemia. Future plans VICONEL offers potential for i) further digitalisation and automation of results sharing at the client, facility, and district/national level, ii) integration of additional clinical and diagnostic data, including HIV comorbidities, and iii) embedding randomised trials. Strengths and limitations ### Competing Interest Statement NDL reports having received travel grants to attend IAS, AIDS, and CROI conferences from Gilead Sciences Sarl and ViiV Healthcare. All other authors declare that they have no competing interests. ### Funding Statement This study was funded by the Swiss National Science Foundation (IZ07Z0\_160876/1, obtained by NDL; PCEFP3\_181355, obtained by NDL) and ESTHER Switzerland (acquired by NDL). JAB receives her salary through grants from Fondation Botnar (REG-19-008, obtained by NDL and JAB) and the University of Basel Research Fund Junior Researchers (3ZX1422, acquired quired by JAB). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The National Health Research Ethics Committee in Lesotho has approved the cohort study (ID 134-2016) and waived consent for analyses using routine data. A biobank consent form for the further use of plasma left over after viral load testing is periodically offered at several sites. Several studies nested within VICONEL but involving non-routine procedures have required a separate study protocol and separate informed consent; data from such studies is not analysed here except insofar as it consists of additional viral load testing or overlaps with routinely collected data. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Aggregated or de-identified individual patient data can be shared upon reasonable request and signing of a data sharing agreement. Investigators must submit a concept sheet detailing the required data and planned analyses to the corresponding author for internal review and approval. Publications arising from VICONEL data are subject to written approval by the Sponsor/Chief Investigator and/or the Principal Investigator. VICONEL investigators or contributors shall be co-authors on publications using VICONEL data, provided they fulfil authorship criteria as defined by the International Committee of Medical Journal Editors.

Numerous mammalian viruses are routinely analyzed in clinical diagnostic laboratories around the globe or serve as indispensable model systems in viral research. Potentially infectious viral entities are handled as blood, biopsies, or cell and tissue culture samples. Countless protocols describe methods for virus fixation and inactivation, yet for many, a formal proof of safety and completeness of inactivation remains to be shown. While modern nucleic acid extraction methods work quite effectively, data are largely lacking on possible residual viral infectivity, e.g., when assessed after extended culture times, which maximizes the sensitivity for low levels of residual infectiousness. Therefore, we examined the potency and completeness of inactivation procedures on virus-containing specimens when applying commonly used fixatives like formaldehyde or nucleic acid extraction/lysis buffers. Typical representatives of different virus classes, including RNA and DNA viruses, enveloped and non-enveloped, such as adenovirus, enterovirus, lentivirus, and coronavirus, were used, and the reduction in the in vitro infectiousness was assessed for standard protocols. Overall, a 30-minute incubation with formaldehyde at room temperature effectively inactivated all tested enveloped and non-enveloped viruses. Full inactivation of HIV-1 and ECHO-11 was also achieved with all buffers in the test, whereas for SARS-CoV-2 and AdV-5, only five of the seven lysis buffers were fully effective under the tested conditions.

Reverse genetic systems enable the engineering of RNA virus genomes and are instrumental in studying RNA virus biology. With the recent outbreak of the coronavirus disease 2019 pandemic, already established methods were challenged by the large genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Herein we present an elaborated strategy for the rapid and straightforward rescue of recombinant plus-stranded RNA viruses with high sequence fidelity using the example of SARS-CoV-2. The strategy called CLEVER (CLoning-free and Exchangeable system for Virus Engineering and Rescue) is based on the intracellular recombination of transfected overlapping DNA fragments allowing the direct mutagenesis within the initial PCR-amplification step. Furthermore, by introducing a linker fragment – harboring all heterologous sequences – viral RNA can directly serve as a template for manipulating and rescuing recombinant mutant virus, without any cloning step. Overall, this strategy will facilitate recombinant SARS-CoV-2 rescue and accelerate its manipulation. Using our protocol, newly emerging variants can quickly be engineered to further elucidate their biology. To demonstrate its potential as a reverse genetics platform for plus-stranded RNA viruses, the protocol has been successfully applied for the cloning-free rescue of recombinant Chikungunya and Dengue virus.

While combination therapy completely suppresses HIV-1 replication in blood, functional virus persists in CD4(+) T cell subsets in non-peripheral compartments that are not easily accessible. To fill this gap, we investigated tis-sue-homing properties of cells that transiently appear in the circulating blood. Through cell separation and in vitro stimulation, the HIV-1 "Gag and Envelope reactivation co-detection assay"(GERDA) enables sensitive detection of Gag+/Env+ protein-expressing cells down to about one cell per million using flow cytometry. By associating GERDA with proviral DNA and polyA-RNA transcripts, we corroborate the presence and functionality of HIV-1 in critical body compartments utilizing t-distributed stochastic neighbor embedding (tSNE) and density -based spatial clustering of applications with noise (DBSCAN) clustering with low viral activity in circulating cells early after diagnosis. We demonstrate transcriptional HIV-1 reactivation at any time, potentially giving rise to intact, infectious particles. With single-cell level resolution, GERDA attributes virus production to lymph -node-homing cells with central memory T cells (T(CM)s) as main players, critical for HIV-1 reservoir eradication.

Abstract Introduction Monitoring HIV viral load (HVL) in people living with HIV (PLHIV) on antiretroviral therapy (ART) is recommended by the World Health Organization. Implementation of HVL testing programs have been affected by logistic and organizational challenges. Here we describe the HVL monitoring cascade in a rural setting in Tanzania and compare turnaround times (TAT) between an on-site and a referral laboratory. Methods In a nested study of the prospective Kilombero and Ulanga Antiretroviral Cohort (KIULARCO) we included PLHIV aged ≥ 15 years, on ART for ≥ 6 months after implementation of routine HVL monitoring in 2017. We assessed proportions of PLHIV with a blood sample taken for HVL, whose results came back, and who were virally suppressed (HVL < 1000 copies/mL) or unsuppressed (HVL ≥ 1000 copies/mL). We described the proportion of PLHIV with unsuppressed HVL and adequate measures taken as per national guidelines and outcomes among those with low-level viremia (LLV; 100–999 copies/mL). We compare TAT between on-site and referral laboratories by Wilcoxon rank sum tests. Results From 2017 to 2020, among 4,454 PLHIV, 4,238 (95%) had a blood sample taken and 4,177 (99%) of those had a result. Of those, 3,683 (88%) were virally suppressed. In the 494 (12%) unsuppressed PLHIV, 425 (86%) had a follow-up HVL (102 (24%) within 4 months and 158 (37%) had virologic failure. Of these, 103 (65%) were already on second-line ART and 32/55 (58%) switched from first- to second-line ART after a median of 7.7 months (IQR 4.7–12.7). In the 371 (9%) PLHIV with LLV, 327 (88%) had a follow-up HVL. Of these, 267 (82%) resuppressed to < 100 copies/ml, 41 (13%) had persistent LLV and 19 (6%) had unsuppressed HVL. The median TAT for return of HVL results was 21 days (IQR 13–39) at the on-site versus 59 days (IQR 27–99) at the referral laboratory (p < 0.001) with PLHIV receiving the HVL results after a median of 91 days (IQR 36–94; similar for both laboratories). Conclusion Robust HVL monitoring is achievable in remote resource-limited settings. More focus is needed on care models for PLHIV with high viral loads to timely address results from routine HVL monitoring.

Vaccines have been central in ending the COVID-19 pandemic, but newly emerging SARS-CoV-2 variants increasingly escape first-generation vaccine protection. To fill this gap, live particle-based vaccines mimicking natural infection aim at protecting against a broader spectrum of virus variants. We designed “single-cycle SARS-CoV-2 viruses” (SCVs) that lack essential viral genes, possess superior immune-modulatory features and provide an excellent safety profile in the Syrian hamster model. Full protection of all intranasally vaccinated animals was achieved against an autologous challenge with SARS-CoV-2 virus using an Envelope-gene-deleted vaccine candidate. By deleting key immune-downregulating genes, sterilizing immunity was achieved with an advanced candidate without virus spread to contact animals. Hence, SCVs have the potential to induce a broad and durable protection against COVID-19 superior to a natural infection.

The coronavirus disease 2019 (COVID-19), caused by a novel coronavirus (SARS-CoV-2), has spread worldwide, affecting over 250 million people and resulting in over five million deaths. Antivirals that are effective are still limited. The antiviral activities of the Petasites hybdridus CO2 extract Ze 339 were previously reported. Thus, to assess the anti-SARS-CoV-2 activity of Ze 339 as well as isopetasin and neopetasin as major active compounds, a CPE and plaque reduction assay in Vero E6 cells was used for viral output. Antiviral effects were tested using the original virus (Wuhan) and the Delta variant of SARS-CoV-2. The antiviral drug remdesivir was used as control. Pre-treatment with Ze 339 in SARS-CoV-2-infected Vero E6 cells with either virus variant significantly inhibited virus replication with IC50 values of 0.10 and 0.40 μg/mL, respectively. The IC50 values obtained for isopetasin ranged between 0.37 and 0.88 μM for both virus variants, and that of remdesivir ranged between 1.53 and 2.37 μM. In conclusion, Ze 339 as well as the petasins potently inhibited SARS-CoV-2 replication in vitro of the Wuhan and Delta variants. Since time is of essence in finding effective treatments, clinical studies will have to demonstrate if Ze339 can become a therapeutic option to treat SARS-CoV-2 infections.

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract ChAdOx1 nCov-19 and Ad26.COV2.S are approved vaccines inducing protective immunity against SARS-CoV-2 infection in humans by expressing the Spike protein of SARS-CoV-2. We analyzed protein content and protein composition of ChAdOx1 nCov-19 and Ad26.COV2.S by biochemical methods and by mass spectrometry. Four out of four tested lots of ChAdOx1 nCoV-19 contained significantly higher than expected levels of host cell proteins (HCPs) and of free viral proteins. The most abundant contaminating HCPs belonged to the heat-shock protein and cytoskeletal protein families. The HCP content exceeded the 400 ng specification limit per vaccine dose, as set by the European Medicines Agency (EMA) for this vaccine, by at least 25-fold and the manufacturer’s batch-release data in some of the lots by several hundred-fold. In contrast, three tested lots of the Ad26.COV2.S vaccine contained only very low amounts of HCPs. As shown for Ad26.COV2.S production of clinical grade adenovirus vaccines of high purity is feasible at an industrial scale. Correspondingly, purification procedures of the ChAdOx1 nCov-19 vaccine should be modified to remove protein impurities as good as possible. Our data also indicate that standard quality assays, as they are used in the manufacturing of proteins, have to be adapted for vectored vaccines. Editor's evaluation This research shows that a commonly used commercial vaccine for COVID-19 harbors contaminating proteins derived from the human cell line in which it is produced. The health significance of these contaminants (if any) remains unknown. This paper is important because lot purity and processing of vaccines is rarely scrutinized in the scientific realm, and instead is typically analyzed only by the companies themselves. https://doi.org/10.7554/eLife.78513.sa0 Decision letter eLife's review process Introduction Beside mRNA vaccines, adenoviral vector-based vaccines have turned out to be an essential mainstay of the vaccination campaign against COVID-19. The AstraZeneca COVID-19 vaccine (AZD1222, Vaxzevria, ChAdOx1 nCov-19 – short ChAdOx1) is based on Chimpanzee Adenovirus Y25 (Dicks et al., 2012) . The Johnson & Johnson Ad26.COV2.S vaccine is based on Human Adenovirus type 26 (HAdV-D26) (Bos et al., 2020). Both vaccines express the full-length Spike protein of SARS-CoV-2. The ability of adenovirus-based vaccines to induce potent humoral and cellular immune responses and their overall safety record had made them strong candidates to successfully fight the COVID-19 pandemic. This has turned out true, with billions of adenovirus-based vaccine doses administered to vaccinees and with very low rates of serious adverse events observed. In the past, and before the COVID-19 pandemic, adenovirus-based vaccines have not been produced even close at the scale required to supply billions of doses. Thus, for all vaccines including for adenovirus-based vaccines scale-up of production processes in a very short period of time has been a significant challenge. Results Protein impurities detected in the AstraZeneca ChAdOx1 nCov-19 vaccine We initially analyzed three different lots of ChAdOx1 by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) followed by silver staining, comparing the staining pattern of the proteins with those of HAdV-C5-EGFP, an HAdV-C5-based adenovirus vector expressing EGFP, purified by CsCl ultracentrifugation. The vaccine is produced in human T-REx-293 cells to prevent expression of the SARS-CoV-2 spike protein during vector production. The vaccine is then purified by a combination of filtration steps and anion-exchange chromatography (European-Medicines-Agency, 2021a; Fedosyuk et al., 2019). Previously, it has been shown, that simian adenovirus vectors including ChAdOx1 can be purified at high yield and purity using such technology for purification (Fedosyuk et al., 2019). To meet global needs and to enable commercial manufacturing of clinical grade material at a very large scale, some modifications and simplifications, respectively, had been introduced both into the upstream and downstream processes, as has been described in detail (Joe et al., 2022). Although in our experiment the same number of viral particles was loaded, the staining pattern of ChAdOx1 looked very different, when compared to the adenoviral vector control (Figure 1). Figure 1 Download asset Open asset Protein staining of HAdV-C5-EGFP and three ChAdOx1 nCoV-19 vaccine lots. 3 × 109 adenoviral vector particles were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) under denaturing and reducing conditions. Proteins were visualized by silver staining. Known HAdV-C5 proteins are labeled. Three different vaccine lots (ABV4678, ABV5811, and ABV7764) of ChAdOx1, produced by the manufacturer, were analyzed. Figure 1—source data 1 Original file of the full raw unedited gel: Protein staining of HAdV-C5-EGFP and three ChAdOx1 nCov-19 vaccine lots. https://cdn.elifesciences.org/articles/78513/elife-78513-fig1-data1-v2.pdf Download elife-78513-fig1-data1-v2.pdf Figure 1—source data 2 Uncropped gel with the relevant bands labeled: Protein staining of HAdV-C5-EGFP and three ChAdOx1 nCov-19 vaccine lots. https://cdn.elifesciences.org/articles/78513/elife-78513-fig1-data2-v2.pdf Download elife-78513-fig1-data2-v2.pdf While staining of HAdV-C5-EGFP proteins resulted in the expected band pattern, representing distinct major capsid proteins of HAdV-C5 including Hexon, Penton Base, IIIa, and Fiber, protein staining of three different lots of ChAdOx1 showed many more bands than could be explained by proteins from viral particles. Additionally, despite loading the same number of particles, band intensities varied between the ChAdOx1 lots. We also analyzed the ChAdOx1 nCoV-19 lots at the DNA level by quantitative polymerase chain reaction (PCR). Results confirmed the exclusive presence of viral DNA, while genomic DNA of the host cell was not detected (data not shown). Detection of substantial amounts of host cell proteins in the vaccine To determine the protein composition of the vaccine, we performed mass spectrometry (MS) analyses from either tryptic in-solution digest directly from the vaccine (Figure 2) or from tryptic in-gel-digest after SDS–PAGE, with similar results (Figure 2—figure supplement 1 and Figure 2—source data 1). Figure 2 with 2 supplements see all Download asset Open asset Distribution of proteins in three ChAdOx1 nCov-19 vaccine lots. The protein composition of three lots of ChAdOx1 (ABV4678, ABV5811, and ABV7764) was analyzed by mass spectrometry following in-solution protein digest. Spectral data were aligned via search engine with human and viral databases (Figure 2—source data 1). (A) Percentage of total intensities associated with proteins from the respective organism were subsequently summed. (B) Percentage of structural and nonstructural adenoviral proteins detected in ChAdOx1 nCov-19 vaccine lot ABV5811. (C) Intensity distribution of the top 20 proteins of the ChAdOx1 nCov-19 vaccine lot ABV5811 detected. Proteins that originate from Homo sapiens are depicted in black; proteins that originate from ChAdOx1 are depicted in blue-gray; *nonstructural adenoviral proteins. Figure 2—source data 1 List of proteins detected in three ChAdOx1 nCov-19 vaccine lots by mass spectrometry. https://cdn.elifesciences.org/articles/78513/elife-78513-fig2-data1-v2.xlsx Download elife-78513-fig2-data1-v2.xlsx Based on intensity comparisons of liquid chromatography/mass spectrometry (LC/MS) signals, we estimate that in lot ABV5811 about 70% of the detected protein content was of human and only 30% of virus origin. In lots ABV4678 and ABV7764 approximately 50% of detected proteins were of human origin (Figure 2A). Beside the expected structural viral proteins forming the virion (Hexon, Penton base, IIIa, Fiber, V, VI, VII, VIII, IX, and others) also several nonstructural viral proteins were detected at high abundancy, although they are not part of the mature viral particle (Figure 2B,C and Figure 2—figure supplement 2). To the detected nonstructural viral proteins belong, for example, the 100K protein, a multifunctional scaffolding protein involved in the trimerization of Hexon, and the DNA-binding protein, which plays an essential role in the replication of the viral genome during infection. In lot ABV5811 nonstructural proteins represented 30% of detected adenoviral proteins (Figure 2B). Since in the assembly process during virus propagation only a part of the available viral capsid proteins is used for particle formation, we assume that in the vaccine product also significant amounts of structural proteins were present as monomers, oligomers, or incomplete viral capsid assemblies. Not being part of the mature viral particle, both nonstructural viral proteins and nonencapsidated structural proteins are referred to as product-related impurities. Peptides from more than 1000 different human proteins, derived from the human production cell line, were detected (Figure 2—source data 1). They were derived from different cellular compartments including cytoplasm, nucleus, endoplasmic reticulum, Golgi apparatus, and others. Relative amounts of human versus viral proteins were variable between lots (Figure 2A), as was already assumed based on the silver-stained gels (Figure 1). Among the human proteins found in the vaccine and beside several cytoskeletal proteins including Vimentin, Tubulin, Actin, and Actinin, the group of heat-shock proteins (HSPs) and chaperones stood out in abundancy. Among the top abundant proteins, HSP 90-beta and HSP 90-alpha as cytosolic HSPs (9.5% and 4.3% of the total proteins, respectively) and three chaperones of the endoplasmic reticulum (transitional endoplasmic reticulum ATPase, Endoplasmin, and Calreticulin) were present (Figure 2C and Figure 2—figure supplement 2). Proteins from Bos taurus from fetal calf serum used for growth of T-REx-293 producer cells, Spike protein of SARS-CoV-2 and T-Rex-293 cell-derived proteins (E1B from HAdV-C5, Tet-Repressor) were detected at low or negligible levels (Figure 2—source data 1). Lack of host cell proteins in the Ad26.COV2.S vaccine A second approved adenoviral vaccine, based on HAdV-D26, is also purified using filtration and chromatography steps (European-Medicines-Agency, 2021b). To our knowledge, more detailed information on the purification process is not publicly available. We compared again a CsCl-purified HAdV-C5-EGFP adenovirus vector with three lots of Ad26.COV2.S (lots 21C10-01, XD955, XE395) and a further (fourth) lot of ChAdOx1 (ABV9317) (Figure 3) and found very little protein contamination in Ad26.COV2.S compared to ChAdOx1 (Figure 3A-C and Figure 3—source data 1). Figure 3 with 2 supplements see all Download asset Open asset Comparison of Ad26.COV2.S and ChAdOx1 nCov-19 vaccines by biochemical and proteomic analysis. (A) Proteins corresponding to each 3 × 109 vector particles of three lots of Ad26.COV2.S (21C10-01, XD955, and XE395), one lot of ChAdOx1 (ABV9317) and CsCl-purified HAdV-C5-EGFP (control), as indicated, were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) performed under denaturing and reducing conditions. Proteins were visualized by silver staining. Known HAdV-C5 proteins are indicated. Note that on SDS–PAGE the stained protein band corresponding to Penton Base of HAdV-C5 (571 amino acids, predicted molecular weight [MW]: 63.4 kDa) has a slower migration behavior than expected, likely due to a flexible loop that is structurally disordered in HAdV-C5 (Flatt et al., 2013) and known to be hypervariable in different adenovirus types (Zubieta et al., 2005). Compared to HAdV-C5, in Chimpanzee Adenovirus Y25 (532 amino acids, MW: 532 kDa) and in HAdV-D26 (519 amino acids, MW: 58.6) this loop is reduced in size and, according to the PONDR-Fit online tool (Xue et al., 2010), less disordered (data not shown), explaining, why in Ad26.COV2.S and in ChAdOx1 the Penton Base-corresponding signals likely overlap with those of the smaller viral proteins IIIa or fiber. (B–D) Ad26.COV2.S (lot XD955) and ChAdOx1 nCov-19 (lot ABV9317) were analyzed by mass spectrometry following in-solution protein digest. Spectral data were aligned via search engine with human and viral proteins (Figure 3—source data 1). Human proteins are depicted in black; proteins that originate from ChAdOx1 are depicted in blue-gray; proteins that originate from Ad26.COV2.S are depicted in green. (B) Percentage of total intensities of proteins from the respective organism detected in the Ad26.COV2.S (lot XD955) and ChAdOx1 nCov-19 (lot ABV9317) vaccines. (C) Intensity distribution of the top 20 proteins of the Ad26.COV2.S vaccine (lot XD955). (D) Percentage of total intensities of structural and nonstructural adenoviral proteins detected in Ad26.COV2.S (lot XD955) and ChAdOx1 nCov-19 (lot ABV9317). Figure 3—source data 1 List of proteins detected by mass spectrometry in three Ad26.COV2.S vaccine lots, ChAdOx1 nCov-19 vaccine lot ABV9317, and HAdV-C5-EGFP. https://cdn.elifesciences.org/articles/78513/elife-78513-fig3-data1-v2.xlsx Download elife-78513-fig3-data1-v2.xlsx Figure 3—source data 2 Original file of the full raw unedited gel: Comparison of Ad26.COV2.S and ChAdOx1 nCov-19 vaccines. https://cdn.elifesciences.org/articles/78513/elife-78513-fig3-data2-v2.pdf Download elife-78513-fig3-data2-v2.pdf Figure 3—source data 3 Uncropped gel with the relevant bands labeled: Protein staining of HAdV-C5-EGFP, three Johnson&Johnson Ad25-COV2.S vaccine lots and one AstraZeneca ChAdOx1 nCov-19 vaccine lot. https://cdn.elifesciences.org/articles/78513/elife-78513-fig3-data3-v2.pdf Download elife-78513-fig3-data3-v2.pdf Mass spectrometry confirmed that more than 93% of proteins detected in HAdV-C5-EGFP were of HAdV-C5 origin (Figure 3—figure supplement 1). Furthermore, 98% of proteins detected in Ad26.COV2.S (lot XD955) were of HAdV-D26 and less than 1% of human origin. In contrast, in lot ABV9317 of ChAdOx1 70% of proteins were of human and only 30% of adenovirus origin (Figure 3B,C and Figure 3—figure supplement 2). Determination of structural and nonstructural viral proteins revealed viral structural to nonstructural protein ratios of 92% versus 8% and of 70% versus 30% in Ad26.COV2.S compared to ChAdOx1, respectively (Figure 3D). The top human host cell proteins (HCPs) in Ad26.COV2.S, although present only at very low abundancy, were histones and ribosomal proteins, rather than the HSPs and cytoskeletal proteins found in ChAdOx1 (Figure 3—source data 1). Quantification of HCPs present in different ChAdOx1 vaccine lots According to European Medicines Agency (EMA) quality specifications for ChAdOx1, 400 ng is the maximally allowable HCP content per single dose (5 × 1010 vector particles). Since the quantification of total protein amounts in inhomogeneous protein mixtures is inherently afflicted with uncertainty due to differences in biophysical properties of the individual proteins, we used three standard methods to determine the HCP content in the tested ChAdOx1 lots (Table 1). Table 1 Amount of protein impurities per vaccine dose in the ChAdOx1 nCoV-19 vaccine. European Medicines Agency (EMA) quality specifications of accepted host cell protein (HCP) amounts in the ChAdOx1 nCoV-19 vaccine and batch-release data by the manufacturer, obtained through the German information freedom act. Total protein content per dose as determined by three different methods of lots ABV4678, ABV5811, ABV7764, and ABV9317 after subtraction of 12.8 µg virus protein contained in 5 × 1010 vector particles Sweeney and Hennessey, 2002. n = 3. ChAdOx1 nCov-19lot #EMA specification[µg HCP/dose]Batch-release data manufacturer[µg HCP/dose]NanoDrop absorbance 280 nm[µg HCP/dose]Bradford assay[µg HCP/dose]ImageJ analysis[µg HCP/dose]ABV4678≤0.40.0539.0312.369.85ABV5811≤0.40.3114.5324.7824.9ABV7764≤0.40.048974.3729.6612.81ABV9317≤0.4n.a.154.5342.4873.16 We took into account the 12.8 µg protein amount contained in 5 × 1010 vector particles, that can be calculated based on the known 150 MDa molecular weight of adenovirus particles (Sweeney and Hennessey, 2002). As expected, and as apparent from Table 1, total protein contents varied with the method used for analysis. However, independent of the method used and as already expected from inspection of Figure 3A, protein amounts contained in the vaccine significantly exceeded the 400 ng specification limit by a factor of 25 or higher, and the manufacturer’s batch-release data by an even much larger (several hundred-fold) extent. In vivo study to analyze potential effects of process- and product-related impurities on anti-Spike immune responses To test whether the process- and product-related impurities in the ChAdOx1 nCov-19 vaccine might influence immune responses against the encoded SARS-CoV-2 Spike protein upon vaccination, we performed an in vivo immunization experiment in mice. We amplified ChAdOx1 nCov-19 in HEK293T cells and purified this vector to more than 90% purity (renamed UUlm ChAdOx1 nCov-19) by standard CsCl density gradient ultracentrifugation (Figure 4A, Figure 4—figure supplement 1, Figure 4—source data 1). BALB/c mice were immunized by intramuscular injection of 1 × 109 vector particles of AstraZeneca ChAdOx1 nCov-19 (ABV9317) or UUlm ChAdOx1 nCov-19. Two weeks later, spike protein-specific T-cell responses (Figure 4B) and spike protein-specific total antibody titers (Figure 4C) were determined. Figure 4 with 1 supplement see all Download asset Open asset Analysis of effects of impurities in the ChAdOx1 nCoV-19 vaccine on anti-Spike immune responses in vivo. BALB/c mice were injected intramuscularly with phosphate-buffered saline (PBS) or 1 × 109 vector particles of AstraZeneca ChAdOx1 nCoV-19 vaccine (ABV9317) or UUlm ChAdOx1 nCov-19 dissolved in PBS. Fourteen days later, mice were sacrificed and plasma and spleen samples collected. n = 6/group. (A) 3 × 109 adenoviral vector particles of AstraZeneca ChAdOx1 nCoV-19 vaccine (ABV9317) or UUlm ChAdOx1 were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) under denaturing and reducing conditions. Proteins were visualized by silver staining. (B) SARS-CoV-2 spike protein-directed T-cell responses were measured by stimulation of isolated T cells with spike peptide-loaded dimer CoVKd-268 GYLQPRTFL in duplicates and subsequent intracellular staining of INFγ. Results are given as mean % INFγ-positive cells of CD8-positive cells of single animals. (C) SARS-CoV-2 spike protein-specific antibody titers were determined by enzyme-linked immunosorbent assay (ELISA). Purified full-length spike protein was coated and serial plasma dilutions were added in duplicates. Results are given as mean response units (RU) of single animals at representative plasma dilutions of 1:500. n.s.: statistically not significant. Figure 4—source data 1 List of proteins detected by mass spectrometry in UUlm ChAdOx1 nCov-19. https://cdn.elifesciences.org/articles/78513/elife-78513-fig4-data1-v2.xlsx Download elife-78513-fig4-data1-v2.xlsx Figure 4—source data 2 Original file of the full raw unedited gel: Protein staining of ChAdOx1 nCov-19 vaccine lot and UUlm ChAdOx1. https://cdn.elifesciences.org/articles/78513/elife-78513-fig4-data2-v2.pdf Download elife-78513-fig4-data2-v2.pdf Figure 4—source data 3 Uncropped gel with the relevant bands labeled: Protein staining of ChAdOx1 nCoV-19 vaccine lot and UUlm ChAdOx1. https://cdn.elifesciences.org/articles/78513/elife-78513-fig4-data3-v2.pdf Download elife-78513-fig4-data3-v2.pdf T-cell responses (against one tested T-cell epitope) were 1.3-fold lower after vaccination using UUlm ChAdOx1 nCov-19 compared to vaccination using AstraZeneca ChAdOx1 nCov-19. Similarly, total antibody titers were also 1.3-fold lower after vaccination using UUlm ChAdOx1 nCov-19 compared to vaccination using AstraZeneca ChAdOx1 nCov-19. However, differences between the two groups were not statistically significant in both parameters, indicating that in this limited study performed in mice there was no indication for obvious interference in induced immune responses related to the HCPs in the original AstraZeneca ChAdOx1 nCov-19 vaccine. Detection of ATPase activity in the ChAdOx1 nCov-19 vaccine Since HSPs and chaperones, found at substantial levels in the ChAdOx1 nCov-19 vaccine are equipped with ATPase activity and because ADP is a strong activator of platelets, we analyzed, whether ATPase activity was detectable in the ChAdOx1 nCov-19 vaccine. ChAdOx1 nCov-19 (lot ABV9317) was incubated with ATP and the hydrolysis of ATP to ADP was determined using a commercial assay. Results confirmed the presence of ATPase activity in the vaccine (Figure 5A,B). Figure 5 Download asset Open asset Analysis of ATPase activity in the ChAdOx1 nCoV-19 vaccine. Conversion of ATP into ADP by ATPase activity of ChAdOx1 nCoV-19 vaccine lot ABV9317 was analyzed using a fluorescence-based commercially available ADP2 FI Assay kit, showing increasing fluorescence intensity signals with increasing amounts of ADP. Negative controls: 4-[2-hydroxyethyl]-1-piperazineethanesulfonic acid (HEPES) instead of ChAdOx1 nCov-19, no addition of ATP; Positive control: addition of ADP instead of ATP. (A) Heatmap of fluorescence intensity of Alexa594 fluorophore. (B) Quantification of measured fluorescence intensity. RFU = response fluorescence units. n = 3. Discussion When analyzing the protein composition of ChAdOx1, a vaccine with an overall good safety record and with high efficacy in preventing or ameliorating COVID-19, we found that the HCP content in ChAdOx1 exceeded the 400 ng/dose specification limit, as set by the EMA, by at least 25-fold in all tested lots, in some of the lots substantially more (Table 1). Compared to the manufacturer’s batch-release data the difference between actual and assumed HCP content, respectively, was even several hundred-fold in some of the lots. The substantial lot-to-lot variation in HCP content in a total of four tested samples (Figure 1 and Table 1) also indicated a lack of robustness of the process used for purification of the vaccine, a process, which recently has been described in detail (Joe et al., 2022). In contrast, in three tested lots of the Ad26.COV2.S vaccine, contamination with HCPs was negligible, confirming that excellent purification of clinical grade adenovirus vaccines is possible at an industrial scale. One can assume that the vast majority of the more than 1000 different proteins detected, most of which present at low abundancy, will not cause any adverse effects. We note that the most abundant proteins do not belong to the group of ‘high risk’ HCPs such as cytokines, proteases, and lipases, as they have been described in the context of protein therapeutics (Jones et al., 2021). Whether or not some of the detected human and/or viral protein contaminants might enhance early clinical vaccine reactions such as flu-like symptoms, very often observed within 1 or 2 days after intramuscular injection of vaccines, is a question that cannot be easily addressed. There is a possibility that some of the proteins, in particular those with higher abundancy, might be more than inert bystanders. For example, extracellular HSPs are known to modulate innate and adaptive immune responses, can exacerbate preexisting inflammatory condition, have been associated with autoimmunity and can even become targets auf autoimmune responses themselves (Binder, 2014; Routsias and Tzioufas, 2006; Tamura et al., 2016). They very efficiently initiate specific immune responses by receptor-mediated uptake of HSP–peptide complexes in antigen-presenting cells (APCs), mainly via CD91 and scavenger receptors (Binder, 2014; Binder et al., 2000). Since the HSPs present in the vaccine are derived from T-REx-293 cells, in principle they could mediate the transfer to APCs of peptides derived from the 293 cell source, of autologous peptides from vaccinated individuals and also of viral proteins. Like many viruses, adenovirus has been reported to induce HSPs during amplification in production cells (Santoro et al., 2010), likely to accommodate for a need in help by HSPs in the folding and production of large amounts of structural and nonstructural viral proteins and in virion assembly. Thus, virus infection-mediated induction of HSPs could explain the abundancy of HSPs in the vaccine product in case of insufficient purification. Because HSPs are endowed with ATPase activity, we searched for and detected ATPase activity in ChAdOx1 (Figure 5), raising the possibility that local ADP generation in the ATP-rich skeletal muscle tissue after injection might contribute to activation of platelets following ADP receptor binding. Shortly after receiving conditional marketing authorization in Europe, cases of thrombotic thrombocytopenia were observed, an unusual and, if untreated, often fatal syndrome of thrombosis of the cerebral venous sinuses and other large vessels together with low platelet counts. Occurring between 5 and 28 days after vaccination (Othman et al., 2021), this syndrome, now coined vaccine-induced immune thrombotic thrombocytopenia (VITT) or thrombosis with thrombocytopenia syndrome, and related to autoimmune heparin-induced thrombocytopenia (Greinacher et al., 2017), was found to be linked to vaccination with ChAdOx1 (Paul-Ehrlich-Institut, 2021), and has been described to be mediated by platelet-activating antibodies against platelet factor 4 (PF4) (Greinacher et al., 2021a ; Greinacher et al., 2021b; Pavord et al., 2021; Schultz et al., 2021; Scully et al., 2021; Tiede et al., 2021; Wolf et al., 2021). Also Ad26.COV2.S has been linked to VITT (Muir et al., 2021; Paul-Ehrlich-Institut, 2021), however at a lower frequency (Paul-Ehrlich-Institut, 2022). It has recently been speculated that impurities in ChAdOx1 could be causally involved in the pathogenesis of VITT (Greinacher et al., 2021a). Since VITT has been found associated with both ChAdOx1 and Ad26.COV2.S, at a lower frequency in the latter (Paul-Ehrlich-Institut, 2021), and because, according to our data, ChAdOx1 but not Ad26.COV2.S contained high amounts of human and free viral proteins, our findings support the notion that the protein impurities present in ChAdOx1 at least are not the primary trigger for the induction of VITT. Incidentally, PF4 itself was not found in our analyses as a contaminant. Whether the impurities might increase the likelihood and frequency of its occurrence in case of ChAdOx1, for example by serving as an inflammatory or immunological cofactor remains a possibility. Interestingly, epidemiologic studies have indicated that RNA vaccines are associated with thromboembolic events but not with VITT (Paul-Ehrlich-Institut, 2021). We speculate that differences in duration and/or levels of Spike protein expression, as to be expected in adenoviral vectors versus mRNA vaccines, might also be a relevant factor in the pathogenesis of VITT, as well as the secretion of the Spike protein due to aberrant splicing events, as recently observed with adenovirus-based vaccines, more so with ChAdOx1 than with Ad26.COV2.S (Kowarz et al., 2022). However, it is important to stress that the frequency of severe thromboembolic events occurring after natural infection with SARS-CoV-2 is much higher than after vaccination with any of the vaccines in use including ChAdOx1 and Ad26.COV2.S (Lau et al., 2021; Nopp et al., 2020). We also considered that intramuscular injection of contaminating free viral or nonviral proteins might influence the quality of the immune response and potentially affect activity and efficacy of the vaccine. In a pilot in vivo immunization experiment performed in mice, anti-Spike antibody and T-cell responses were not significantly altered following immunization with ChAdOx1 compared to an identical vector without HCP contamination (Figure 4), suggesting that the protein impurities did not interfere with the induction of immune responses against the Spike antigen. However, we acknowledge that our study was limited by testing recognition of only a single T-cell epitope. In the biopharmaceutical industry, the removal of HCPs from the biological product is a critical quality attribute, since residual HCPs pose a risk to patient safety (Vanderlaan et al., 2018; Wang et al., 2009; Zhu-Shimoni et al., 2014). The main risks generally discussed relate either to the biological activity of the contaminant, to effects of the impurities enhancing immune responses against the therapeutic protein (antidrug antibodies) or to inducing antibodies against the impurities (Vanderlaan et al., 2018). These concerns are relevant as they have been observed clinically. Overall, problems with HCPs are rare events and classical protein biopharmaceuticals have an excellent safety record (de Zafra et al., 2015). In cases, in which problems have been observed, therapeutic proteins have been produced in heterologous (i.e., nonhuman) production systems such as E. coli, yeast, and Chinese Hamster Ovary (CHO) cell lines (de Zafra et al., 2015). For example, antidrug antibodies were generated in patients against human growth hormone (Vanderlaan et al., 2018), produced in E. coli, apparently due to a contaminating protein that had been missed by an unsuitable HCP assay. Another case, more closely re

Background: Children living with HIV and taking antiretroviral therapy (ART) are a priority group for routine viral load (VL) monitoring. As per Lesotho guidelines, a VL >= 1000 copies/mL ("unsuppressed") should trigger adherence counseling and a follow-up VL; 2 consecutive unsuppressed VLs ("virologic failure") qualify for switching to second-line ART, with some exceptions. Here, we describe the pediatric VL cascade in Lesotho. Methods: In a prospective open cohort study comprising routine VL results from 22 clinics in Lesotho, we assessed outcomes along the VL cascade for children who had at least 1 VL test from January 2016 through June 2020. Data were censored on February 10, 2021. Results: In total, 1215 children received 5443 VL tests. The median age was 10 years (interquartile range 7-13) and 627/1215 (52%) were female; 362/1215 (30%) had at least 1 unsuppressed VL. A follow-up VL was available for 325/362 (90%), although only for 159/362 (44%) within 6 months of the first unsuppressed VL. Of those with a follow-up VL, 172/329 (53%) had virologic failure and 123/329 (37%) qualified for switching to second-line ART. Of these, 55/123 (45%) were ever switched, although only 9/123 (7%) were switched within 12 weeks of the follow-up VL. Delays were more pronounced in rural facilities. Overall, 100/362 (28%) children with an unsuppressed VL received a timely follow-up VL and, if required, a timely regimen switch. Conclusions: Despite access to VL monitoring, clinical management was suboptimal. HIV programs should prioritize timely clinical action to maximize the benefits of VL monitoring.

Objectives: Phylogenetic analyses of 2 or more countries allow to detect differences in transmission dynamics of local HIV-1 epidemics beyond differences in demographic characteristics. Methods: A maximum-likelihood phylogenetic tree was built using pol-sequences of the Swiss HIV Cohort Study (SHCS) and the Austrian HIV Cohort Study (AHIVCOS), with international background sequences. Three types of phylogenetic cherries (clusters of size 2) were analyzed further: (1) domestic cherries; (2) international cherries; and (3) SHCS/AHIVCOS-cherries. Transmission group and ethnicities observed within the cherries were compared with the respective distribution expected from a random distribution of patients on the phylogeny. Results: The demographic characteristics of the AHIVCOS (included patients: 3 ' 141) and the SHCS (included patients: 12 ' 902) are very similar. In the AHIVCOS, 36.5% of the patients were in domestic cherries, 8.3% in international cherries, and 7.0% in SHCS/AHIVCOS cherries. Similarly, in the SHCS, 43.0% of the patients were in domestic cherries, 8.2% in international cherries, and 1.7% in SHCS/AHIVCOS cherries. Although international cherries in the SHCS were dominated by heterosexuals with men who have sex with men being underrepresented, the opposite was the case for the AHIVCOS. In both cohorts, cherries with one patient belonging to the transmission group intravenous drug user and the other one non-intravenous drug user were underrepresented. Conclusions: In both cohorts, international HIV transmission plays a major role in the local epidemics, mostly driven by men who have sex with men in the AHIVOS, and by heterosexuals in the SHCS, highlighting the importance of international collaborations to understand global HIV transmission links on the way to eliminate HIV.