ABSTRACT
In this study we profiled vaccine-induced polyclonal antibodies as well as plasmablast-derived mAbs from individuals who received SARS-CoV-2 spike mRNA vaccine. Polyclonal antibody responses in vaccinees were robust and comparable to or exceeded those seen after natural infection. However, the ratio of binding to neutralizing antibodies after vaccination was greater than that after natural infection and, at the monoclonal level, we found that the majority of vaccine-induced antibodies did not have neutralizing activity. We also found a co-dominance of mAbs targeting the NTD and RBD of SARS-CoV-2 spike and an original antigenic-sin like backboost to spikes of seasonal human coronaviruses OC43 and HKU1. Neutralizing activity of NTD mAbs but not RBD mAbs against a clinical viral isolate carrying E484K as well as extensive changes in the NTD was abolished, suggesting that a proportion of vaccine-induced RBD binding antibodies may provide substantial protection against viral variants carrying single E484K RBD mutations.
Subject(s)
Antibodies, Viral/immunology , COVID-19 Vaccines/immunology , RNA, Messenger/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Vaccination , Amino Acid Substitution , Angiotensin-Converting Enzyme 2/immunology , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/isolation & purification , Antibodies, Neutralizing/immunology , Antibody Formation/immunology , Binding, Competitive , Humans , Immunoglobulin G/metabolism , Mutation/genetics , Protein Domains , Somatic Hypermutation, Immunoglobulin/geneticsABSTRACT
It is thought that mRNA-based vaccine-induced immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) wanes quickly, based mostly on short-term studies. Here, we analyzed the kinetics and durability of the humoral responses to SARS-CoV-2 infection and vaccination using >8,000 longitudinal samples collected over a 3-year period in New York City. Upon primary immunization, participants with pre-existing immunity mounted higher antibody responses faster and achieved higher steady-state antibody titers than naive individuals. Antibody kinetics were characterized by two phases: an initial rapid decay, followed by a stabilization phase with very slow decay. Booster vaccination equalized the differences in antibody concentration between participants with and without hybrid immunity, but the peak antibody titers decreased with each successive antigen exposure. Breakthrough infections increased antibodies to similar titers as an additional vaccine dose in naive individuals. Our study provides strong evidence that SARS-CoV-2 antibody responses are long lasting, with initial waning followed by stabilization.
Subject(s)
COVID-19 , Vaccines , Humans , SARS-CoV-2 , Antibody Formation , Vaccination , Immunization, Secondary , mRNA Vaccines , Antibodies, ViralABSTRACT
Influenza B virus (IBV) infections can cause severe disease in children and the elderly. Commonly used antivirals have lower clinical effectiveness against IBV compared to influenza A viruses (IAV). Neuraminidase (NA), the second major surface protein on the influenza virus, is emerging as a target of broadly protective antibodies that recognize the NA active site of IAVs. However, similarly broadly protective antibodies against IBV NA have not been identified. Here, we isolated and characterized human monoclonal antibodies (mAbs) that target IBV NA from an IBV-infected patient. Two mAbs displayed broad and potent capacity to inhibit IBV NA enzymatic activity, neutralize the virus in vitro, and protect against lethal IBV infection in mice in prophylactic and therapeutic settings. These mAbs inserted long CDR-H3 loops into the NA active site, engaging residues highly conserved among IBV NAs. These mAbs provide a blueprint for the development of improved vaccines and therapeutics against IBVs.
Subject(s)
Antibodies, Viral/immunology , Catalytic Domain/immunology , Influenza B virus/immunology , Neuraminidase/immunology , Viral Proteins/immunology , Animals , Antibodies, Monoclonal/immunology , Cell Line , Dogs , Female , HEK293 Cells , Humans , Influenza A virus/immunology , Influenza, Human/immunology , Leukocytes, Mononuclear/immunology , Madin Darby Canine Kidney Cells , Mice , Mice, Inbred BALB C , Middle Aged , Orthomyxoviridae Infections/immunologyABSTRACT
The Omicron (B.1.1.529) variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was initially identified in November 2021 in South Africa and Botswana, as well as in a sample from a traveller from South Africa in Hong Kong1,2. Since then, Omicron has been detected globally. This variant appears to be at least as infectious as Delta (B.1.617.2), has already caused superspreader events3, and has outcompeted Delta within weeks in several countries and metropolitan areas. Omicron hosts an unprecedented number of mutations in its spike gene and early reports have provided evidence for extensive immune escape and reduced vaccine effectiveness2,4-6. Here we investigated the virus-neutralizing and spike protein-binding activity of sera from convalescent, double mRNA-vaccinated, mRNA-boosted, convalescent double-vaccinated and convalescent boosted individuals against wild-type, Beta (B.1.351) and Omicron SARS-CoV-2 isolates and spike proteins. Neutralizing activity of sera from convalescent and double-vaccinated participants was undetectable or very low against Omicron compared with the wild-type virus, whereas neutralizing activity of sera from individuals who had been exposed to spike three or four times through infection and vaccination was maintained, although at significantly reduced levels. Binding to the receptor-binding and N-terminal domains of the Omicron spike protein was reduced compared with binding to the wild type in convalescent unvaccinated individuals, but was mostly retained in vaccinated individuals.
Subject(s)
Antibodies, Neutralizing/immunology , COVID-19 Vaccines/immunology , COVID-19/immunology , COVID-19/virology , Convalescence , Immune Evasion/immunology , Immune Sera/immunology , SARS-CoV-2/immunology , 2019-nCoV Vaccine mRNA-1273/immunology , Adult , Antibodies, Monoclonal/immunology , Antibodies, Viral/immunology , BNT162 Vaccine/administration & dosage , BNT162 Vaccine/immunology , COVID-19/transmission , Female , Humans , Immunization, Secondary , Models, Molecular , Neutralization Tests , SARS-CoV-2/classification , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunologyABSTRACT
To replicate in their hosts, viruses have to navigate the complexities of the mammalian cell, co-opting mechanisms of cellular physiology while defeating restriction factors that are dedicated to halting their progression. Primate lentiviruses devote a relatively large portion of their coding capacity to counteracting restriction factors by encoding accessory proteins dedicated to neutralizing the antiviral function of these intracellular inhibitors. Research into the roles of the accessory proteins has revealed the existence of previously undetected intrinsic defenses, provided insight into the evolution of primate lentiviruses as they adapt to new species and uncovered new targets for the development of therapeutics. This Review discusses the biology of the restriction factors APOBEC3, SAMHD1 and tetherin and the viral accessory proteins that counteract them.
Subject(s)
Antigens, CD/metabolism , Cytosine Deaminase/metabolism , HIV Infections/immunology , HIV-1/physiology , Host Specificity , Immune Evasion , Monomeric GTP-Binding Proteins/metabolism , Viral Regulatory and Accessory Proteins/metabolism , APOBEC Deaminases , Animals , Biological Evolution , Cytidine Deaminase , GPI-Linked Proteins/metabolism , HIV Infections/virology , Humans , Molecular Targeted Therapy , SAM Domain and HD Domain-Containing Protein 1ABSTRACT
In late 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first detected in China and has since caused a pandemic of coronavirus disease 2019 (COVID-19). The first case of COVID-19 in New York City was officially confirmed on 1 March 2020 followed by a severe local epidemic1. Here, to understand seroprevalence dynamics, we conduct a retrospective, repeated cross-sectional analysis of anti-SARS-CoV-2 spike antibodies in weekly intervals from the beginning of February to July 2020 using more than 10,000 plasma samples from patients at Mount Sinai Hospital in New York City. We describe the dynamics of seroprevalence in an 'urgent care' group, which is enriched in cases of COVID-19 during the epidemic, and a 'routine care' group, which more closely represents the general population. Seroprevalence increased at different rates in both groups; seropositive samples were found as early as mid-February, and levelled out at slightly above 20% in both groups after the epidemic wave subsided by the end of May. From May to July, seroprevalence remained stable, suggesting lasting antibody levels in the population. Our data suggest that SARS-CoV-2 was introduced in New York City earlier than previously documented and describe the dynamics of seroconversion over the full course of the first wave of the pandemic in a major metropolitan area.
Subject(s)
Antibodies, Viral/blood , Antibodies, Viral/immunology , COVID-19 Serological Testing/statistics & numerical data , COVID-19/epidemiology , COVID-19/immunology , Epidemiological Monitoring , SARS-CoV-2/immunology , Adolescent , Adult , Ambulatory Care/statistics & numerical data , COVID-19/diagnosis , COVID-19/virology , Child , Child, Preschool , Cross-Sectional Studies , Female , Humans , Incidence , Infant , Infant, Newborn , Male , Middle Aged , New York City/epidemiology , Spike Glycoprotein, Coronavirus/immunology , Time Factors , Urban Population/statistics & numerical data , Young AdultABSTRACT
Viruses are obligate parasites and thus require the machinery of the host cell to replicate. Inhibition of host factors co-opted during active infection is a strategy hosts use to suppress viral replication and a potential pan-antiviral therapy. To define the cellular proteins and processes required for a virus during infection is thus crucial to understanding the mechanisms of virally induced disease. In this report, we generated fully infectious tagged influenza viruses and used infection-based proteomics to identify pivotal arms of cellular signaling required for influenza virus growth and infectivity. Using mathematical modeling and genetic and pharmacologic approaches, we revealed that modulation of Sec61-mediated cotranslational translocation selectively impaired glycoprotein proteostasis of influenza as well as HIV and dengue viruses and led to inhibition of viral growth and infectivity. Thus, by studying virus-human protein-protein interactions in the context of active replication, we have identified targetable host factors for broad-spectrum antiviral therapies.
Subject(s)
Host-Parasite Interactions/physiology , Influenza A virus/physiology , Influenza A virus/pathogenicity , Models, Theoretical , Virus Replication/physiology , Dengue Virus/pathogenicity , Dengue Virus/physiology , HIV/pathogenicity , HIV/physiology , Humans , Immunoprecipitation , Mass Spectrometry , Protein Folding , ProteomicsABSTRACT
Current influenza virus vaccines have to be closely matched to circulating strains to provide good protection, and antigenic drift and emerging pandemic influenza virus strains present a difficult challenge for them. Universal influenza virus vaccines, including chimeric hemagglutinin (cHA)-based constructs that target the conserved stalk domain of hemagglutinin, are in clinical development. Due to the conservation of the stalk domain, antibodies directed to it show broad binding profiles, usually within group 1 and group 2 influenza A or influenza B virus phylogenies. However, determining the binding breadth of these antibodies with commonly used immunological methods can be challenging. Here, we analyzed serum samples from a phase I clinical trial (CVIA057, NCT03300050) using an influenza virus protein microarray (IVPM). The IVPM technology allowed us to assess immune responses not only to a large number of group 1 hemagglutinins but also group 2 and influenza B virus hemagglutinins. In CVIA057, different vaccine modalities, including a live attenuated influenza virus vaccine and inactivated influenza virus vaccines with or without adjuvant, all in the context of cHA constructs, were tested. We found that vaccination with adjuvanted, inactivated vaccines induced a very broad antibody response covering group 1 hemagglutinins, with limited induction of antibodies to group 2 hemagglutinins. Our data show that cHA constructs do indeed induce very broad immune responses and that the IVPM technology is a useful tool to measure this breadth that broadly protective or universal influenza virus vaccines aim to induce. IMPORTANCE The development of a universal influenza virus vaccine that protects against seasonal drifted, zoonotic, or emerging pandemic influenza viruses would be an extremely useful public health tool. Here, we test a technology designed to measure the breadth of antibody responses induced by this new class of vaccines.
Subject(s)
Cross Reactions , Influenza Vaccines , Influenza, Human , Humans , Adjuvants, Immunologic , Antibodies, Viral , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Influenza B virus , Influenza Vaccines/immunology , Influenza, Human/prevention & control , Influenza A virusABSTRACT
Type I interferons (IFN-Is) are a group of potent inflammatory and antiviral cytokines. They induce IFN stimulated genes (ISGs), which act as proinflammatory mediators, antiviral effectors, and negative regulators of the IFN-I signaling cascade itself. One such regulator is interferon stimulated gene 15 (ISG15). Humans with complete ISG15 deficiency express persistently elevated levels of ISGs, and consequently, exhibit broad spectrum resistance to viral infection. Here, we demonstrate that IFN-I primed fibroblasts derived from ISG15-deficient individuals are more resistant to infection with single-cycle HIV-1 compared to healthy control fibroblasts. Complementation with both wild-type (WT) ISG15 and ISG15ΔGG (incapable of ISGylation while retaining negative regulation activity) was sufficient to reverse this phenotype, restoring susceptibility to infection to levels comparable to WT cells. Furthermore, CRISPR-edited ISG15ko primary CD4+ T cells were less susceptible to HIV-1 infection compared to cells treated with non-targeting controls. Transcriptome analysis of these CRISPR-edited ISG15ko primary CD4+ T cells recapitulated the ISG signatures of ISG15 deficient patients. Taken together, we document that the increased broad-spectrum viral resistance in ISG15-deficiency also extends to HIV-1 and is driven by a combination of T-cell-specific ISGs, with both known and unknown functions, predicted to target HIV-1 replication at multiple steps.
Subject(s)
Cytokines , HIV Infections , HIV-1 , Ubiquitins , Antiviral Agents/pharmacology , Cytokines/genetics , HIV Infections/genetics , Humans , Interferon Type I , Ubiquitins/geneticsABSTRACT
In this study, we utilized a panel of human immunoglobulin (Ig) IgA monoclonal antibodies isolated from the plasmablasts of eight donors after 2014/2015 influenza virus vaccination (Fluarix) to study the binding and functional specificities of this isotype. In this cohort, isolated IgA monoclonal antibodies were primarily elicited against the hemagglutinin protein of the H1N1 component of the vaccine. To compare effector functionalities, an H1-specific subset of antibodies targeting distinct epitopes were expressed as monomeric, dimeric, or secretory IgA, as well as in an IgG1 backbone. When expressed with an IgG Fc domain, all antibodies elicited Fc-effector activity in a primary polymorphonuclear cell-based assay which differs from previous observations that found only stalk-specific antibodies activate the low-affinity FcγRIIIa. However, when expressed with IgA Fc domains, only antibodies targeting the stalk domain showed Fc-effector activity in line with these previous findings. To identify the cause of this discrepancy, we then confirmed that IgG signaling through the high-affinity FcγI receptor was not restricted to stalk epitopes. Since no corresponding high-affinity Fcα receptor exists, the IgA repertoire may therefore be limited to stalk-specific epitopes in the context of Fc receptor signaling.
Subject(s)
Epitopes/immunology , Hemagglutinin Glycoproteins, Influenza Virus/immunology , Immunoglobulin A/immunology , Immunoglobulin Fc Fragments/immunology , Influenza A Virus, H1N1 Subtype/immunology , Adult , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/metabolism , Antibody Affinity , Binding Sites, Antibody , Chick Embryo , Cryoelectron Microscopy , Female , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Hemagglutinin Glycoproteins, Influenza Virus/metabolism , Humans , Influenza Vaccines/immunology , Male , Neutrophils/immunology , Neutrophils/virologyABSTRACT
BACKGROUND: The number of exposures to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and to vaccine antigens affect the magnitude and avidity of the polyclonal response. METHODS: We studied binding and avidity of different antibody isotypes to the spike, the receptor-binding domain (RBD), and the nucleoprotein (NP) of wild-type (WT) and BA.1 SARS-CoV-2 in convalescent, mRNA vaccinated and/or boosted, hybrid immune individuals and in individuals with breakthrough cases during the peak of the BA.1 wave. RESULTS: We found an increase in spike-binding antibodies and antibody avidity with increasing number of exposures to infection and/or vaccination. NP antibodies were detectible in convalescent individuals and a proportion of breakthrough cases, but they displayed low avidity. Omicron breakthrough infections elicited high levels of cross-reactive antibodies between WT and BA.1 antigens in vaccinated individuals without prior infection directed against the spike and RBD. The magnitude of the antibody response and avidity correlated with neutralizing activity against WT virus. CONCLUSIONS: The magnitude and quality of the antibody response increased with the number of antigenic exposures, including breakthrough infections. However, cross-reactivity of the antibody response after BA.1 breakthroughs, was affected by the number of prior exposures.
Subject(s)
Antibodies, Viral , Antibody Affinity , Breakthrough Infections , COVID-19 , SARS-CoV-2 , Animals , Humans , Antibodies, Viral/blood , Antibodies, Viral/immunology , Breakthrough Infections/blood , Breakthrough Infections/immunology , Chlorocebus aethiops , COVID-19/blood , COVID-19/immunology , COVID-19/prevention & control , COVID-19 Serological Testing , SARS-CoV-2/immunology , Vaccination , Vero Cells , BNT162 Vaccine/immunology , BNT162 Vaccine/therapeutic useABSTRACT
Influenza virus neuraminidase (NA)-targeting antibodies are an independent correlate of protection against influenza. Antibodies against the NA act by blocking enzymatic activity, preventing virus release and transmission. As we advance the development of improved influenza virus vaccines that incorporate standard amounts of NA antigen, it is important to identify the antigenic targets of human monoclonal antibodies (mAbs). Here, we describe escape mutants generated by serial passage of A/Netherlands/602/2009 (H1N1)pdm09 in the presence of human anti-N1 mAbs. We observed escape mutations on the head domain of the N1 protein around the enzymatic site (S364N, N369T, and R430Q) and also detected escape mutations located on the sides and bottom of the NA (N88D, N270D, and Q313K/R). This work increases our understanding of how human antibody responses target the N1 protein. IMPORTANCE As improved influenza virus vaccines are being developed, the influenza virus neuraminidase (NA) is becoming an important new target for immune responses. By identifying novel epitopes of anti-NA antibodies, we can improve vaccine design. Additionally, characterizing escape mutations in these epitopes aids in identifying NA antigenic drift in circulating viruses.
Subject(s)
Influenza A Virus, H1N1 Subtype , Influenza Vaccines , Influenza, Human , Antibodies, Monoclonal , Antibodies, Viral/metabolism , Epitopes/immunology , Humans , Influenza A Virus, H1N1 Subtype/enzymology , Influenza A Virus, H1N1 Subtype/genetics , Influenza Vaccines/genetics , Influenza Vaccines/immunology , Influenza, Human/virology , Mutation , Neuraminidase/chemistry , Neuraminidase/genetics , Neuraminidase/immunologyABSTRACT
HIV integrates into the host genome, creating a viral reservoir of latently infected cells that persists despite effective antiretroviral treatment. CD4-positive (CD4+) T cells are the main contributors to the HIV reservoir. CD4+ T cells are a heterogeneous population, and the mechanisms of latency establishment in the different subsets, as well as their contribution to the reservoir, are still unclear. In this study, we analyzed HIV latency establishment in different CD4+ T cell subsets stimulated with interleukin 15 (IL-15), a cytokine that increases both susceptibility to infection and reactivation from latency. Using a dual-reporter virus that allows discrimination between latent and productive infection at the single-cell level, we found that IL-15-treated primary human CD4+ T naive and CD4+ T stem cell memory (TSCM) cells are less susceptible to HIV infection than CD4+ central memory (TCM), effector memory (TEM), and transitional memory (TTM) cells but are also more likely to harbor transcriptionally silent provirus. The propensity of these subsets to harbor latent provirus compared to the more differentiated memory subsets was independent of differential expression of pTEFb components. Microscopy analysis of NF-κB suggested that CD4+ T naive cells express smaller amounts of nuclear NF-κB than the other subsets, partially explaining the inefficient long terminal repeat (LTR)-driven transcription. On the other hand, CD4+ TSCM cells display similar levels of nuclear NF-κB to CD4+ TCM, CD4+ TEM, and CD4+ TTM cells, indicating the availability of transcription initiation and elongation factors is not solely responsible for the inefficient HIV gene expression in the CD4+ TSCM subset. IMPORTANCE The formation of a latent reservoir is the main barrier to HIV cure. Here, we investigated how HIV latency is established in different CD4+ T cell subsets in the presence of IL-15, a cytokine that has been shown to efficiently induce latency reversal. We observed that, even in the presence of IL-15, the less differentiated subsets display lower levels of productive HIV infection than the more differentiated subsets. These differences were not related to different expression of pTEFb, and modest differences in NF-κB were observed for CD4+ T naive cells only, implying the involvement of other mechanisms. Understanding the molecular basis of latency establishment in different CD4+ T cell subsets might be important for tailoring specific strategies to reactivate HIV transcription in all the CD4+ T subsets that compose the latent reservoir.
Subject(s)
CD4-Positive T-Lymphocytes , HIV Infections , Interleukin-15 , Virus Latency , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/virology , HIV Infections/immunology , HIV Infections/virology , HIV-1 , Humans , Interleukin-15/pharmacology , NF-kappa B/metabolism , Proviruses , T-Lymphocyte Subsets/drug effects , T-Lymphocyte Subsets/virologyABSTRACT
The public health burden caused by influenza virus infections is not adequately addressed with existing vaccines and antivirals. Identifying approaches that interfere with human-to-human transmission of influenza viruses remains a pressing need. The importance of neuraminidase (NA) activity for the replication and spread of influenza viruses led us to investigate whether broadly reactive human anti-NA monoclonal antibodies (MAbs) could affect airborne transmission of the virus using the guinea pig model. In that model, infection with recent influenza virus clinical isolates resulted in 100% transmission from inoculated donors to recipients in an airborne transmission setting. Anti-NA MAbs were administered either to the inoculated animals on days 1, 2, and 4 after infection or to the naive contacts on days 2 and 4 after donor infection. Administration of NA-1G01, a broadly cross-reactive anti-NA MAb, to either the donor or recipient reduced transmission of the A/New York City/PV02669/2019 (H1N1) and A/New York City/PV01148/2018 (H3N2) viruses. Administration of 1000-3C05, an anti-N1 MAb, to either the donor or recipient reduced transmission of A/New York City/PV02669/2019 (H1N1) virus but did not reduce transmission of A/New York City/PV01148 (H3N2) virus. Conversely, 229-2C06, an anti-N2 MAb, reduced transmission of A/New York City/PV01148 (H3N2) but did not impact transmission of A/New York City/PV02669/2019 (H1N1) virus. Our work demonstrates that anti-NA MAbs could be further developed into prophylactic or therapeutic agents to prevent influenza virus transmission to control viral spread. IMPORTANCE The burden of influenza remains substantial despite unremitting efforts to reduce the magnitude of seasonal influenza epidemics and prepare for pandemics. Although vaccination remains the mainstay of these efforts, current vaccines are designed to stimulate an immune response against the viral hemagglutinin. Interest in the role immunity against neuraminidase plays in influenza virus infection and transmission has recently surged. Human antibodies that bind broadly to neuraminidases of diverse influenza viruses and protect mice against lethal viral challenge have previously been characterized. Here, we show that three such antibodies inhibit the neuraminidase activity of recent isolates and reduce their airborne transmission in a guinea pig model. In addition to contributing to the accumulating support for incorporating neuraminidase as a vaccine antigen, these findings also demonstrate the potential of direct administration of anti-neuraminidase antibodies to individuals infected with influenza virus and to individuals for postexposure prophylaxis to prevent the spread of influenza virus.
Subject(s)
Antibodies, Viral/therapeutic use , Neuraminidase/immunology , Orthomyxoviridae Infections/prevention & control , Viral Proteins/immunology , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/therapeutic use , Antibodies, Viral/immunology , Cross Reactions , Guinea Pigs , Humans , Immunization, Passive , Influenza A Virus, H1N1 Subtype/immunology , Influenza A Virus, H3N2 Subtype/immunology , Influenza, Human/immunology , Orthomyxoviridae Infections/transmissionABSTRACT
In 2022 the World Health Organization declared a Public Health Emergency for an outbreak of mpox, the zoonotic Orthopoxvirus (OPV) affecting at least 104 nonendemic locations worldwide. Serologic detection of mpox infection is problematic, however, due to considerable antigenic and serologic cross-reactivity among OPVs and smallpox-vaccinated individuals. In this report, we developed a high-throughput multiplex microsphere immunoassay using a combination of mpox-specific peptides and cross-reactive OPV proteins that results in the specific serologic detection of mpox infection with 93% sensitivity and 98% specificity. The New York State Non-Vaccinia Orthopoxvirus Microsphere Immunoassay is an important tool to detect subclinical mpox infection and understand the extent of mpox spread in the community through retrospective analysis.
Subject(s)
Mpox (monkeypox) , Orthopoxvirus , Humans , Retrospective Studies , Asymptomatic Infections , Biological Assay , Cross ReactionsABSTRACT
Monkeypox (MPOX) is a zoonotic disease that affects humans and other primates, resulting in a smallpox-like illness. It is caused by monkeypox virus (MPXV), which belongs to the Poxviridae family. Clinically manifested by a range of cutaneous and systemic findings, as well as variable disease severity phenotypes based on the genetic makeup of the virus, the cutaneous niche and respiratory mucosa are the epicenters of MPXV pathogenicity. Herein, we describe the ultrastructural features of MPXV infection in both human cultured cells and cutaneous clinical specimens collected during the 2022-2023 MPOX outbreak in New York City that were revealed through electron microscopy. We observed typical enveloped virions with brick-shaped morphologies that contained surface protrusions, consistent with the classic ultrastructural features of MPXV. In addition, we describe morpho-functional evidence that point to roles of distinct cellular organelles in viral assembly during clinical MPXV infection. Interestingly, in skin lesions, we found abundant melanosomes near viral assembly sites, particularly in the vicinity of mature virions, which provides further insight into virus-host interactions at the subcellular level that contribute to MPXV pathogenesis. These findings not only highlight the importance of electron microscopic studies for further investigation of this emerging pathogen but also in characterizing MPXV pathogenesis during human infection.
Subject(s)
Mpox (monkeypox) , Skin Diseases , Animals , Humans , Monkeypox virus/genetics , Virulence , Primates , GenomicsABSTRACT
Diagnosis by rapid antigen tests (RATs) is useful for early initiation of antiviral treatment. Because RATs are easy to use, they can be adapted for self-testing. Several kinds of RATs approved for such use by the Japanese regulatory authority are available from drug stores and websites. Most RATs for COVID-19 are based on antibody detection of the SARS-CoV-2 N protein. Since Omicron and its subvariants have accumulated several amino acid substitutions in the N protein, such amino acid changes might affect the sensitivity of RATs. Here, we investigated the sensitivity of seven RATs available in Japan, six of which are approved for public use and one of which is approved for clinical use, for the detection of BA.5, BA.2.75, BF.7, XBB.1, and BQ.1.1, as well as the delta variant (B.1.627.2). All tested RATs detected the delta variant with a detection level between 7500 and 75 000 pfu per test, and all tested RATs showed similar sensitivity to the Omicron variant and its subvariants (BA.5, BA.2.75, BF.7, XBB.1, and BQ.1.1). Human saliva did not reduce the sensitivity of the RATs tested. Espline SARS-CoV-2 N showed the highest sensitivity followed by Inspecter KOWA SARS-CoV-2 and V Trust SARS-CoV-2 Ag. Since the RATs failed to detect low levels of infectious virus, individuals whose specimens contained less infectious virus than the detection limit would be considered negative. Therefore, it is important to note that RATs may miss individuals shedding low levels of infectious virus.
Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , COVID-19/diagnosis , Amino Acid Substitution , Antiviral AgentsABSTRACT
Monkeypox virus (MPXV) is a zoonotic orthopoxvirus within the Poxviridae family. MPXV is endemic to Central and West Africa. However, the world is currently witnessing an international outbreak with no clear epidemiological links to travel or animal exposure and with ever-increasing numbers of reported cases worldwide. Here, we evaluated and validated a new, sensitive, and specific real-time PCR-assay for MPXV diagnosis in humans and compare the performance of this novel assay against a Food & Drug Administration-cleared pan-Orthopox RT-PCR assay. We determined specificity, sensitivity, and analytic performance of the PKamp™ Monkeypox Virus RT-PCR assay targeting the viral F3L-gene. In addition, we further evaluated MPXV-PCR-positive specimens by viral culture, electron microscopy, and viral inactivation assays. The limit of detection was established at 7.2 genome copies/reaction, and MPXV was successfully identified in 20 clinical specimens with 100% correlation against the reference method with 100% sensitivity and specificity. Our results demonstrated the validity of this rapid, robust, and reliable RT-PCR assay for specific and accurate diagnosis of MPXV infection in human specimens collected both as dry swabs and in viral transport media. This assay has been approved by NYS Department of Health for clinical use.
Subject(s)
Monkeypox virus , Mpox (monkeypox) , Animals , Humans , Monkeypox virus/genetics , Mpox (monkeypox)/epidemiology , Reverse Transcriptase Polymerase Chain Reaction , Nucleic Acid Amplification Techniques/methods , Real-Time Polymerase Chain ReactionABSTRACT
N6-methyladenosine (m6A) is the most abundant HIV RNA modification but the interplay between the m6A reader protein YTHDF3 and HIV replication is not well understood. We found that knockout of YTHDF3 in human CD4+ T-cells increases infection supporting the role of YTHDF3 as a restriction factor. Overexpression of the YTHDF3 protein in the producer cells reduces the infectivity of the newly produced viruses. YTHDF3 proteins are incorporated into HIV particles in a nucleocapsid-dependent manner permitting the m6A reader protein to limit infection in the new target cell at the step of reverse transcription. Importantly, HIV protease cleaves the virion-incorporated full-length YTHDF3 protein, a process which is blocked by HIV protease inhibitors used to treat HIV infected patients. Mass-spectrometry confirmed the proteolytic processing of YTHDF3 in the virion. Thus, HIV protease cleaves the virion-encapsidated host m6A effector protein in addition to the viral polyproteins to ensure optimal infectivity of the mature virion.
Subject(s)
HIV Protease/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Adenosine/analogs & derivatives , Adenosine/genetics , Adenosine/metabolism , Antiviral Agents/metabolism , CD4-Positive T-Lymphocytes/metabolism , CD4-Positive T-Lymphocytes/virology , HEK293 Cells , HIV Infections/virology , HIV Protease/physiology , HIV-1/genetics , Humans , Primary Cell Culture , Virion/metabolismABSTRACT
The coronavirus disease-2019 (COVID-19) pandemic is still challenging public health systems worldwide, particularly with the emergence of novel SARS-CoV-2 variants with mutations that increase their transmissibility and immune escape. This is the case of the variant of concern Omicron that rapidly spread globally. Here, using epidemiological and genomic data we compared the situations in South Africa as the epicenter of emergence, United Kingdom, and with particular interest New York City. This rapid global dispersal from the place of first report reemphasizes the high transmissibility of Omicron, which needed only two weeks to become dominant in the United Kingdom and New York City. Our analyses suggest that as SARS-CoV-2 continues to evolve, global authorities must prioritize equity in vaccine access and continued genomic surveillance. Future studies are still needed to fully unveil the biological properties of Omicron, but what is certain is that vaccination, large-scale testing, and infection prevention efforts are the greatest arsenal against the COVID-19 pandemic.