Objective This study investigates the prevalence of human pegivirus (HPgV) among SARS-CoV-2-positive individuals within the broader context of viral co-infections that may modulate COVID-19 outcomes. We further assess whether HPgV co-infection is associated with COVID-19 severity. HPgV is a widely circulating but rarely monitored human virus with documented immunomodulatory effects in other viral infections, including HIV and Ebola. While HPgV prevalence is low in the general U.S. population (1–2%), it rises markedly in the setting of chronic viral co-infections, particularly HIV (15–40%). Given its immunologic effects and persistence, HPgV represents a biologically plausible but unexplored viral co-factor SARS-CoV-2 infection. Methods We analyzed four cohorts: SARS-CoV-2–positive individuals, ICU patients with respiratory symptoms but SARS-CoV-2–negative, HIV-positive individuals as a positive control for HPgV detection, and uninfected controls. Results HPgV prevalence among COVID-19 patients was low (2.1%) and comparable to population estimates. As expected, HPgV prevalence was substantially higher in the HIV cohort (34%), validating assay performance and cohort stratification. Among HPgV-positive COVID-19 cases, most experienced mild disease, with directional trends toward reduced severity despite high baseline risk factors. Healthcare workers in the control group showed unexpectedly elevated HPgV prevalence (9.6%). Conclusions HPgV is an unmonitored but widely circulating viral co-infection in humans that may influence host responses to SARS-CoV-2. Although limited by small sample size, these findings are descriptive and hypothesis-generating, supporting further investigation of HPgV and other immunomodulatory viral co-infections in COVID-19. Larger studies will be required to determine whether the observed patterns are reproducible and clinically meaningful.

N6-methyladenosine (m6A) modifications of human immunodeficiency virus type 1 (HIV-1) and cellular RNA contribute to regulation of viral and host gene expression. RNA m6A dysregulation has been implicated in multiple cancers. However, the role of m6A modifications in HIV-1-associated cancers remains unclear. Here, we aim to address this important question using clinical samples. Using enzyme-linked immunosorbent assay (ELISA), RNA m6A levels were quantified in peripheral blood mononuclear cells (PBMCs) from 43 de-identified people living with HIV-1 (PLWH), comparing those with cancer (n = 15) to those without cancer (n = 28). Correlation analyses were performed to evaluate the associations between m6A levels and HIV-1 RNA copies, CD4+ T-cell counts, and age of PLWH. The mRNA and protein expression of m6A regulatory genes was measured by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot, respectively. Furthermore, quantitative transcriptomic profiling of 84 type I interferon (IFN-I)-responsive genes was performed using an RT-qPCR array. We observed that HIV-1 viral load in the cancer group was higher than the non-cancer group. m6A levels of PBMCs were 2.8-fold higher in the cancer group and correlated with the expression of some m6A regulatory genes and proteins. Higher m6A levels were positively associated with HIV-1 RNA copies and negatively associated with CD4+ T-cell counts and age. Transcriptomic analysis of 84 IFN-I-responsive genes revealed upregulation of many pro-inflammatory and interferon-stimulated genes, along with downregulation of some genes in PLWH with cancer. Our findings suggest that HIV-1 infection and cancer-mediated m6A reprogramming may contribute to multifaceted chronic immune activation and malignancy in PLWH. Our results also highlight a post-transcriptional mechanism linking HIV-1 persistence to cancer risk.

Objective:This study investigates the prevalence of human pegivirus (HPgV) in SARS-CoV-2-positive patients within the context of viral co-infections that may modulate COVID-19 outcomes and assesses whether HPgV co-infection is associated with COVID-19 severity. HPgV is a widely circulating but rarely monitored human virus with documented immunomodulatory effects in other viral infections, including HIV and Ebola. While HPgV prevalence is low in the general U.S. population (1-2%), it rises markedly in the setting of chronic viral co-infections, particularly HIV (15-40%). Given its immunologic effects and persistence, HPgV represents a biologically plausible but unexplored viral co-factor SARS-CoV-2 infection. Methods:We analyzed four cohorts: SARS-CoV-2-positive individuals, ICU patients with respiratory symptoms but SARS-CoV-2-negative, HIV-positive individuals as a positive control for HPgV detection, and uninfected controls. Results:HPgV prevalence in COVID-19 patients was low (2.1%) and comparable to population estimates. As expected, HPgV prevalence was substantially higher in the HIV cohort (34%), validating assay performance and cohort stratification. Among HPgV-positive COVID-19 cases, most experienced mild disease, with directional trends toward reduced severity despite high baseline risk factors. Healthcare workers in the control group showed unexpectedly elevated HPgV prevalence (9.6%). Conclusions:HPgV is an unmonitored but widely circulating viral co-infection in humans that may influence host responses to SARS-CoV-2. Although limited by small numbers, our findings support further investigation of HPgV and other immunomodulatory viral co-infections in COVID-19. This study suggests that HPgV co-infection may influence COVID-19 outcomes, warranting further investigation. Highlights:Systematic screening of human pegivirus (HPgV), an unmonitored viral co-infection, in COVID-19 (n = 634).HPgV prevalence in COVID-19 mirrored population estimates but was markedly enriched in HIV (positive control).HPgV-positive COVID-19 cases showed trends toward milder clinical outcomes.Findings highlight the potential relevance of immunomodulatory viral co-infections in SARS-CoV-2 infection. Executive Summary:This study evaluated the prevalence of human pegivirus (HPgV) co-infection among SARS-CoV-2-positive patients as part of a broader effort to understand how concurrent viral infections may influence COVID-19 severity. HPgV is a largely unmonitored, persistent human virus with well-described immunomodulatory effects in other viral infections, yet it has not been systematically evaluated in COVID-19. We therefore screened HPgV prevalence across COVID-19 cases, comparator cohorts, and an HIV-positive cohort as a positive control due to the well-established high prevalence of HPgV in HIV infection. Our findings indicate that HPgV prevalence in SARS-CoV-2-positive and -negative hospitalized individuals are consistent with the general U.S. population range (approximately 1-5%). Healthcare professionals exhibited a higher HPgV prevalence (∼10%), suggesting that repeated occupational viral exposures may influence infection rates. While limited by small numbers, HPgV co-infection in COVID-19 cases was associated with directional trends toward reduced disease severity, warranting further longitudinal and mechanistic investigation. Editor’s summary:This study identifies human pegivirus as a widely circulating, unmonitored viral co-infection in COVID-19 with potential relevance to disease severity.

BACKGROUND:The Randomized Trial to Prevent Vascular Events in HIV (REPRIEVE) found a 36% reduction in major adverse cardiovascular events (MACE) with pitavastatin in people with HIV. Little is known about the relationship between lipid lowering and MACE in this population. We evaluated pitavastatin effects on lipids and examined mediation of pitavastatin effects on MACE through lipid lowering. METHODS:REPRIEVE was a randomised, double-blind, placebo-controlled phase 3 trial evaluating pitavastatin (4 mg daily) or placebo for prevention of MACE in people with HIV. Participants aged 40-75 years, on stable combination antiretroviral therapy (ART), and at low-to-moderate atherosclerotic cardiovascular disease risk with minimally elevated LDL were followed up for a median of 5·6 years. Centrally tested fasting lipids were captured at entry and annually thereafter. The primary MACE outcome, reported previously, was time to first MACE. Here, we report secondary outcomes on fasting lipids. Linear mixed-effects models were used for assessment of the pitavastatin effect on lipids, Cox regression for relationship of lipids to MACE, and Vansteelandt method for mediation analysis. FINDINGS:REPRIEVE enrolled 7769 participants from March 26, 2015, to July 31, 2019, in 12 countries across five Global Burden of Diseases super-regions. The median baseline LDL was 108 mg/dL, and similar across treatment groups. Pitavastatin effects were primarily observed on LDL, with a modest reduction in triglycerides and no apparent effect on HDL. Based on the longitudinal data modelling, the estimated treatment group difference in LDL at month 12 (pitavastatin minus placebo) was -30 mg/dL (95% CI -31 to -29), corresponding to a 30% reduction. The estimated risk of LDL of ≥100 mg/dL at month 12 was 0·18 in the pitavastatin group and 0·57 in the placebo group (relative risk 0·32, 95% CI 0·30-0·34). A 30% lower time-updated average LDL was associated with 20% lower risk of primary MACE (hazard ratio 0·80, 95% CI 0·68-0·94). Of the pitavastatin effect on MACE, 68% was estimated to be mediated through LDL, although with low precision (95% CI 15-574). INTERPRETATION:LDL is strongly related to MACE, and LDL lowering should be an important goal of primary cardiovascular prevention in people with HIV, even in those with minimally elevated LDL. Treatment should aim to achieve accepted primary care prevention targets for LDL. FUNDING:US National Institutes of Health, Kowa Pharmaceuticals America, Gilead Sciences, and ViiV Healthcare.

The Flaviviridae are a family of non-segmented positive-sense enveloped RNA viruses containing significant pathogens including hepatitis C virus and yellow fever virus. Recent large-scale metagenomic surveys have identified many diverse RNA viruses related to classical orthoflaviviruses and pestiviruses but quite different genome lengths and configurations, and with a hugely expanded host range that spans multiple animal phyla, including molluscs, cnidarians and stramenopiles,, and plants. Grouping of RNA-directed RNA polymerase (RdRP) hallmark gene sequences of flavivirus and 'flavi-like' viruses into four divergent clades and multiple lineages within them was congruent with helicase gene phylogeny, PPHMM profile comparisons, and comparison of RdRP protein structure predicted by AlphFold2. These results support their classification into the established order, Amarillovirales, in three families (Flaviviridae, Pestiviridae, and Hepaciviridae), and 14 genera. This taxonomic framework informed by RdRP hallmark gene evolutionary relationships provides a stable reference from which major genome re-organisational events can be understood.

Semen-derived extracellular vesicles (SEVs) have been shown to inhibit transactivation of the long terminal repeat (LTR) in human immunodeficiency virus type 1 (HIV-1, or HIV) and, hence, viral replication by blocking the interaction of the virus’s transcriptional activator Tat and host transcription factors NF-κB and Sp1. The ability of SEVs to regulate the activities of transcription factors suggests that SEVs may contain transcription activators and repressors. Here, we identified host proteins in human SEVs that interacted with the Tat and NF-κB subunit p65. Integrative network and pathway enrichment analyses of these complexes revealed associations with an array of biological functions regulating genome transcription. In particular, several proteins in SEVs could bind to both Tat and NF-κB: the scaffolding and cell signaling regulatory protein AKAP9, the G protein signaling regulator ARHGEF28, the small nuclear RNA processor INTS1, the epigenetic reader BRD2, and the transcription elongation inhibitor NELFB. NF-κB p65–bound NELFB also interacted with HEXIM1, another transcription elongation inhibitor, suggesting that SEVs may inhibit HIV propagation through networks of transcriptional regulation and repression. One Sentence Summary Proteins in vesicles shed from human semen may repress HIV by targeting transcription factors. ### Competing Interest Statement The authors have declared no competing interest. National Institutes of Health, , R01DA042348-01, R01DA050169, R21/R33DA053643 Veterans Affairs Iowa, , BX000207

Extracellular particles (EPs), an umbrella term encompassing membrane-enclosed extracellular vesicles (EVs) and non-vesicular extracellular particles ([NVEPs], previously described as extracellular condensates [ECs]) contain a complex cargo of biomolecules, including DNA, RNA, proteins, and lipids, reflecting the physiological state of their cell of origin. Identifying proteins associated with EPs that regulate host responses to physiological and pathophysiological processes is of critical importance. Here, we report the findings of our study to gain insight into the proteins associated with NVEPs. We used samples from human semen, the rat brain, and the rhesus macaque (RM) brain and blood to assess the physical properties and proteome profiles of NVEPs from these specimens. The results show significant differences in the zeta potential, concentration, and size of NVEPs across different species. We identified 938, 51, and 509 total proteins from NVEPs isolated from rat brain tissues, RM blood, and human seminal plasma, respectively. The species-specific protein networks show distinct biological themes, while the species-conserved protein interactome was identified with six proteins (ALB, CST3, FIBA/FGA, GSTP1, PLMN/PLG, PPIA) associated with NVEPs in all samples. The six NVEP-associated proteins are prone to aggregation and formation of wide, insoluble, unbranched filaments with a cross-beta sheet quaternary structure, such as amyloid fibrils. Protein-to-function analysis indicates that the six identified proteins are linked to the release of dopamine, immune-mediated inflammatory disease, replication of RNA viruses, HIV-HCV co-infection, and inflammation. These interesting findings have created an opportunity to evaluate NVEPs for their potential use as biomarkers of health and disease. Additional in-depth studies are needed to clarify when and how these proteins sustain their physiological role or transition to pathogenic roles.

BACKGROUND:Dengue virus serotype 2 and 3 (DENV-2 and -3) infections are associated with more severe disease outcomes than DENV-1 and 4, though a biological explanation for this has not been identified. METHODS:DENV serotype effects on human T-cell activation were assessed by measuring T-cell receptor (TCR)-mediated interleukin 2 release following TCR stimulation. DENV envelope (env) proteins were expressed in Jurkat CD4+ T-cell lines, and regions inhibiting TCR inhibition were mapped by mutagenesis. TCR signaling pathway inhibition was characterized by total internal reflection fluorescence microscopy and immunoblot. Reverse genetics validated env mapping results. RESULTS:T-cell incubation with DENV-1 and -4 inhibited TCR signaling, whereas DENV-2 and -3 did not. Inhibition did not require viral replication. DENV env protein expression inhibited TCR by interfering with activation of proximal TCR signaling events. Amino acids (aa's) 49 to 62 of DENV 1 env were sufficient to inhibit TCR signaling, with env aa's 55 and 66 being critical. Reverse genetics confirmed that substitution of DENV-2 and -3 aa's 55 and 66 into DENV-1 and -4 reversed TCR inhibition and that DENV-1 aa's 55 and 66 introduced into DENV-2 and -3 enabled TCR inhibition. CONCLUSIONS:DENV-2 and -3 are associated with more severe clinical disease than DENV-1 and -4; however, no biological explanation for this difference has been previously identified. We found that DENV-1 and -4 viral particles and env proteins blunt T-cell responses by interfering with proximal TCR signaling, while DENV-2 and -3 do not, potentially explaining DENV pathogenic outcomes in primary and secondary infection.

The persistence of HIV latent reservoir is the major challenge to HIV cure because latent viruses serve as sources for viral rebound upon ART cessation. Mechanisms regulating viral persistence are not well understood; thus, there is a compelling need for research focusing on addressing the knowledge gap related to HIV persistence. The present study focuses on the effect of extracellular condensates (ECs) on latent HIV/SIV reactivation in the brain in the context of HIV infection using the SIV-infected rhesus macaque model. We used in vitro model systems of post-integration latency and primary peripheral blood mononuclear cells isolated from HIV-infected ART-suppressed donors to explore the role of basal ganglia (BG) isolated extracellular condensates (ECs) in reprogramming HIV latent cells. We found that BG ECs from uninfected macaques (VEH) and SIV infected macaques (VEH | SIV) activated latent HIV transcription in various model systems. VEH | SIV ECs significantly increased the expression and production of viral antigen in latently infected cells. Activation of viral transcription, antigen expression, and latency reactivation was inhibited by ECs from the brain of macaques treated with Delta-9-tetrahydrocannabinol (THC) and infected with SIV (THC | SIV). Virus produced by latently infected cells treated with VEH | SIV ECs potentiated cell-cell and cell-free HIV transmission. VEH | SIV ECs also reversed dexamethasone-mediated inhibition of HIV transcription while TNFα-mediated reactivation of latency was reversed by THC | SIV ECs. Transcriptome and secretome analyses of total RNA and supernatants from latently infected cells treated with ECs revealed significant alterations in gene expression and cytokine secretion. THC | SIV ECs increased secretion of Th2 and decreased secretion of proinflammatory cytokines. Most strikingly, while VEH/SIV ECs robustly induced expression of HIV RNA in latently HIV-infected cells, increased the frequency of HIV gag p24 expressing cells in HIV-infected CD4 + T cells within PBMCs, and production of extracellular HIV gag p24, long-term low-dose THC administration enriched ECs with anti-inflammatory cargo that significantly diminished their ability to reactivate latent HIV, an indication that ECs are endogenous host factors that may regulate HIV persistence.

Replication of HIV-1 requires the coordinated action of host and viral transcription factors, most critically the viral transactivator Tat and the host nuclear factor κB (NF-κB). This activity is disrupted in infected cells that are cultured with extracellular vesicles (EVs) present in human semen, suggesting that they contain factors that could inform the development of new therapeutics. Here, we explored the contents of semen-derived EVs (SEVs) from uninfected donors and individuals with HIV-1 and identified host proteins that interacted with HIV Tat and the NF-κB subunit p65. Integrative network and pathway enrichment analyses of these complexes revealed associations with an array of biological functions regulating gene expression. Several proteins in SEVs bound to both Tat and NF-κB p65: the scaffolding and cell signaling regulatory protein AKAP9, the G protein signaling regulator ARHGEF28, the epigenetic reader BRD2, the small nuclear RNA processor INTS1, and the transcription elongation inhibitor NELFB. When complexed with p65, NELFB also interacted with HEXIM1, another transcription elongation inhibitor, suggesting that SEVs may inhibit HIV-1 propagation through multiple networks of transcriptional activation and repression. Exploring these data and the underlying mechanisms may inform the development of more effective or more durable therapeutics against HIV.

Flaviviridae is a family of non-segmented positive-sense RNA viruses that includes major pathogens such as hepatitis C virus, dengue viruses and yellow fever virus. Recent large-scale metagenomic surveys have identified many RNA viruses related to members of this family, such as orthoflaviviruses and pestiviruses. These viruses diverge by having different genome lengths and configurations, and host range. Here we performed an analysis of RNA-directed RNA polymerase (RdRP) hallmark gene sequences of flaviviruses and 'flavi-like' viruses. We uncovered four divergent clades and multiple lineages that are congruent with phylogenies of their helicase genes, protein profile hidden Markov model profiles, and evolutionary relationships based on predicted RdRP protein structures. These results support their classification into three families (Flaviviridae, Pestiviridae and Hepaciviridae) and 12 genera in the established order Amarillovirales, with groupings correlating with genome properties and host range. This taxonomy provides a framework for future evolutionary studies on this important viral family.

Introduction Pandemic influenza vaccine development focuses on the hemagglutinin (HA) antigen for potency and immunogenicity. Antibody responses targeting the neuraminidase (NA) antigen, or the HA stalk domain have been implicated in protection against influenza. Responses to the NA and HA-stalk domain following pandemic inactivated influenza are not well characterized in humans. Material and methods In a series of clinical trials, we determine the vaccines' NA content and demonstrate that NA inhibition (NAI) antibody responses increase in a dose-dependent manner following a 2-dose priming series with AS03-adjuvanted influenza A(H7N9) inactivated vaccine (A(H7N9) IIV). NAI antibody responses also increase with interval extension of the 2-dose priming series or following a 5-year delayed boost with a heterologous adjuvanted A(H7N9) IIV. Neither concomitant seasonal influenza vaccination given simultaneously or sequentially, nor use of heterologous A(H7N9) IIVs in the 2-dose priming series had an appreciable effect on NAI antibody responses. Anti-HA stalk antibody responses were minimal and not durable. Conclusions We provide evidence for strategies to improve anti-neuraminidase responses which can be further standardized for pandemic preparedness. Clinical Trial Registry Numbers NCT03312231, NCT03318315, NCT03589807, NCT03738241.

Background:All Food and Drug Administration-approved laboratory-based human immunodeficiency virus (HIV) screening tests have high sensitivity and specificity. There is an HIV diagnostic test algorithm recommended by the Centers for Disease Control and Prevention (CDC) and Association of Public Health Laboratories (APHL) in the United States. Still, diagnostic challenges can occur in real-world practice. We describe an unusual case that exhibited false positive results for HIV screening and confirmation testing. Case Summary:A 27-year-old cisgender female had HIV testing as part of antenatal care. Her local HIV-1/2 antigen/antibody (Ag/Ab) screening test was reactive, and the HIV Ab differentiation assay was positive for HIV-1. Using CDC and APHL algorithm guidelines, she was diagnosed with HIV-1 infection. However, her HIV-1 ribonucleic acid (RNA) quantitative polymerase chain reaction was undetected. Although she immigrated from an HIV-1 high prevalence region, she was otherwise at low risk for HIV transmission. Qualitative HIV-1 deoxyribonucleic acid/RNA was undetected, and HIV-1 Western blot analysis was only positive for HIV-1 gp160 reactivity, confirming that her initial testing represented a false positive and that she did not have HIV infection. The rest of her pregnancy and delivery course was uneventful. Conclusion:Our case highlights the limitations of current HIV diagnostic tests and challenges in interpretation. Clinicians should be aware that false-positive tests are rare and be reminded to consider the pre-test probability when interpreting results.

Background: Human infections with the avian influenza A(H7N9) virus were first reported in China in 2013 and continued to occur in annual waves. In the 2016/2017 fifth wave, Yangtze River Delta (YRD) lineage viruses, which differed antigenically from those of earlier waves, predominated. Methods: In this phase 2 double-blinded trial we randomized 720 adults >= 19 years of age to receive two injections of a YRD lineage inactivated A/Hong Kong/125/2017 fifth-wave H7N9 vaccine, given 21 days apart, at doses of 3.75, 7.5, and 15 mu g of hemagglutinin (HA) with AS03A adjuvant and at doses of 15 and 45 mu g of HA without adjuvant. Results: Two doses of adjuvanted vaccine were required to induce HA inhibition (HI) antibody titers > 40 in most participants. After two doses of the 15 mu g H7N9 formulation, given with or without AS03 adjuvant, the proportion achieving a HI titer >= 40 against the vaccine strain at 21 days after the second vaccination was 65 % (95 % CI, 57 %-73 %) and 0 % (95 % CI, 0 %-4%), respectively. Among those who received two doses of the 15 mu g adjuvanted formulation the proportion with HI titer >= 40 at 21 days after the second vaccination was 76 % (95 % CI, 66 %-84 %) in those 19-64 years of age and 49 % (95 % CI, 37 %-62 %) in those >= 65 years of age. Responses to the adjuvanted vaccine formulations did not vary by HA content. Antibody responses declined over time and responses against drifted H7N9 strains were diminished. Overall, the vaccines were well tolerated but, as expected, adjuvanted vaccines were associated with more frequent solicited systemic and local adverse events. Conclusions: AS03 adjuvant improved the immune responses to an inactivated fifth-wave H7N9 influenza vaccine, particularly in younger adults, but invoked lower responses to drifted H7N9 strains. These findings may inform future influenza pandemic preparedness strategies.

BACKGROUND:Live attenuated vaccines alter immune functions and are associated with beneficial outcomes. We previously demonstrated that live attenuated yellow fever virus (YFV) vaccine (LA-YF-Vax) dampens T-cell receptor (TCR) signaling in vitro via an RNA-based mechanism. We examined study participants before and after LA-YF-Vax to assess TCR-mediated functions in vivo. METHODS:Serum samples and peripheral blood mononuclear cells (PBMCs) were obtained before and after LA-YF-Vax (with or without additional vaccines) or quadrivalent influenza vaccine. TCR-mediated activation was determined by interleukin 2 release or phosphorylation of the lymphocyte-specific Src kinase. TCR-regulating phosphatase (protein tyrosine phosphatase receptor type E [PTPRE]) expression was also measured. RESULTS:Compared with prevaccination findings, LA-YF-Vax recipient PBMCs demonstrated transient reduction in interleukin 2 release after TCR stimulation and PTPRE levels, unlike in control participants who received quadrivalent influenza vaccine. YFV was detected in 8 of 14 participants after LA-YF-Vax. After incubation of healthy donor PBMCs in serum-derived extracellular vesicles prepared from LA-YF-Vax recipients, TCR signaling and PTPRE levels were reduced after vaccination, even in participants without detectable YFV RNA. CONCLUSIONS:LA-YF-Vax reduces TCR functions and PTPRE levels after vaccination. Extracellular vesicles from serum recapitulated this effect in healthy cells. This likely contributes to the reduced immunogenicity for heterologous vaccines after LA-YF-Vax administration. Identification of specific immune mechanisms related to vaccines should contribute to understanding of the "off-target," beneficial effects of live vaccines.

aCenter for HIV and Hepatogastroenterology, Duesseldorf, Germany bThe Iowa City Veterans Healthcare System and the University of Iowa, Iowa City, Iowa cJohns Hopkins School of Medicine, Baltimore, Maryland, USA. Correspondence to Stefan Mauss, MD, Center for HIV and Hepatogastroenterology, Humboldt Strasse 18, Duesseldorf 40237, Germany. Tel: +49 211 239 552 21; fax: +49 211 239 552 60; e-mail: [email protected] Received 7 March, 2024 Accepted 19 March, 2024

There is a critical need to understand the effectiveness of serum elicited by different SARS-CoV-2 vaccines against SARS-CoV-2 variants. We describe the generation of reference reagents comprised of post-vaccination sera from recipients of different primary vaccines with or without different vaccine booster regimens in order to allow standardized characterization of SARS-CoV-2 neutralization in vitro. We prepared and pooled serum obtained from donors who received a either primary vaccine series alone, or a vaccination strategy that included primary and boosted immunization using available SARS-CoV-2 mRNA vaccines (BNT162b2, Pfizer and mRNA-1273, Moderna), replication-incompetent adenovirus type 26 vaccine (Ad26.COV2·S, Johnson and Johnson), or recombinant baculovirus-expressed spike protein in a nanoparticle vaccine plus Matrix-M adjuvant (NVX-CoV2373, Novavax). No subjects had a history of clinical SARS-CoV-2 infection, and sera were screened with confirmation that there were no nucleocapsid antibodies detected to suggest natural infection. Twice frozen sera were aliquoted, and serum antibodies were characterized for SARS-CoV-2 spike protein binding (estimated WHO antibody binding units/ml), spike protein competition for ACE-2 binding, and SARS-CoV-2 spike protein pseudotyped lentivirus transduction. These reagents are available for distribution to the research community (BEI Resources), and should allow the direct comparison of antibody neutralization results between different laboratories. Further, these sera are an important tool to evaluate the functional neutralization activity of vaccine-induced antibodies against emerging SARS-CoV-2 variants of concern.ImportanceThe explosion of COVID-19 demonstrated how novel coronaviruses can rapidly spread and evolve following introduction into human hosts. The extent of vaccine- and infection-induced protection against infection and disease severity is reduced over time due to the fall in concentration, and due to emerging variants that have altered antibody binding regions on the viral envelope spike protein. Here, we pooled sera obtained from individuals who were immunized with different SARS-CoV-2 vaccines and who did not have clinical or serologic evidence of prior infection. The sera pools were characterized for direct spike protein binding, blockade of virus-receptor binding, and neutralization of spike protein pseudotyped lentiviruses. These sera pools were aliquoted and are available to allow inter-laboratory comparison of results and to provide a tool to determine the effectiveness of prior vaccines in recognizing and neutralizing emerging variants of concern.

ABSTRACT Approximately 15% of the global human population is viremic with human pegivirus (HPgV), a +ssRNA virus in the Flaviviridae family. An unusual feature of HPgV is its ability to persistently infect individuals without causing overt disease or evoking robust immune responses, but this phenomenon is poorly understood due to a dearth of systems for studying HPgV. In this study, we create the first mouse model of PgV infection by adapting a PgV discovered in a wild rat (RPgV) to infect the standard laboratory mouse. Adaptation to the mouse initially required defective innate immunity and the accumulation of a single mutation in the E2 envelope glycoprotein, but passage into wild-type (WT) mice resulted in twelve additional mutations that enable persistent high-titer viremia, closely recapitulating the course of HPgV in humans. Mouse-adapted (ma)PgV infection of various knockout mice showed that lymphocytes exert a significant antiviral effect in the chronic phase of infection, but that this effect is also unable to fully control viremia in most individuals. Chronic type-I interferon signaling appears to paradoxically enable maPgV persistence, likely via T cell dysfunction that has been demonstrated in other chronic viral infections. However, unlike many persistent viruses, maPgV does not depend upon the induction of PD-1-mediated immune tolerance to maintain persistence. In-depth analysis of rare WT mice that achieved sterilizing maPgV immunity suggests that multiple possible paths to achieving PgV immunity exist and may include a combination of cellular, humoral, and non-canonical mechanisms. Altogether, our creation of maPgV opens up the vast murine toolkit for understanding the enigmatic biology of PgVs. In addition to novel insights into multiple aspects of PgV immunity, the lack of PD-1-mediated immune tolerance induced by PgV infection is unique among persistent viruses and suggests a highly novel mechanism of immune evasion. AUTHOR SUMMARY Viruses capable of persistently infecting an individual host have developed sophisticated mechanisms for evading host immunity, and understanding these mechanisms can reveal novel features of the host immune system. One such virus, human pegivirus (HPgV), infects ∼15% of the global human population, but little is known about its biology beyond the fact that it does not cause overt disease. We created the first mouse model of PgV infection by adapting a rat pegivirus to infect laboratory mice. This mouse-adapted virus (maPgV) caused infection that was detectable in the blood of mice for >300 days without causing signs of disease, closely recapitulating the course of HPgV in humans. This enabled unprecedented exploration of PgV immunity, revealing a pro-viral role for type-I interferon in chronic infection; a lack of PD-1-mediated tolerance to PgV infection; and multiple mechanisms by which PgV immunity can be achieved by an immunocompetent host. These data indicate that the PgV immune evasion strategy has aspects that are both common and unique among persistent viral infections. The creation of maPgV represents the first PgV infection model in wild-type mice, thus opening the entire toolkit of the mouse host to enable further investigation of persistent RNA infections.