SARS-CoV-2 mouse adaptation selects virulence mutations that cause TNF-driven age-dependent severe disease with human correlates.

The diversity of COVID-19 disease in otherwise healthy people, from seemingly asymptomatic infection to severe life-threatening disease, is not clearly understood. We passaged a naturally occurring near-ancestral SARS-CoV-2 variant, capable of infecting wild-type mice, and identified viral genomic mutations coinciding with the acquisition of severe disease in young adult mice and lethality in aged animals. Transcriptomic analysis of lung tissues from mice with severe disease elucidated a host antiviral response dominated mainly by interferon and IL-6 pathway activation in young mice, while in aged animals, a fatal outcome was dominated by TNF and TGF-ß signaling. Congruent with our pathway analysis, we showed that young TNF-deficient mice had mild disease compared to controls and aged TNF-deficient animals were more likely to survive infection. Emerging clinical correlates of disease are consistent with our preclinical studies, and our model may provide value in defining aberrant host responses that are causative of severe COVID-19.

CRISPR-Cas13b-mediated suppression of hepatitis B virus replication and protein expression.

BACKGROUND & AIMS: New antiviral approaches are urgently required that target multiple aspects of the hepatitis B virus (HBV) replication cycle to improve rates of functional cure. HBV RNA represents a novel therapeutic target. Here, we programmed Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas13b endonuclease, to specifically target the HBV pregenomic RNA (pgRNA) and viral mRNAs in a novel approach to reduce HBV replication and protein expression. METHODS: Cas13b CRISPR RNAs (crRNAs) were designed to target multiple regions of HBV pgRNA. Mammalian cells with replication competent wildtype HBV DNA of different genotypes, a HBV stable cell line, a HBV infection model and a hepatitis B surface antigen (HBsAg)-expressing stable cell line were transfected with PspCas13b-blue fluorescent protein (BFP) and crRNAs plasmids and the impact on HBV replication and protein expression was measured. WT HBV DNA, PspCas13b-BFP and crRNA plasmids were simultaneously hydrodynamically injected into mice, and sera HBsAg was measured. PspCas13b mRNA and crRNA were also delivered by lipid nanoparticles (LNP) in a HBsAg-expressing stable cell line and the impact on secreted HBsAg determined. RESULTS: Our HBV targeting crRNAs strongly suppressed HBV replication and protein expression in mammalian cells by up to 96% (p<0.0001). HBV protein expression was also reduced in an HBV stable cell line and in the HBV infection model. CRISPR-Cas13b crRNAs reduced HBsAg expression by 50% (p<0.0001) in vivo. LNP-encapsulated PspCas13b mRNA reduced secreted HBsAg by 87% (p=0.0168) in a HBsAg-expressing stable cell line. CONCLUSIONS: Together, these results show that CRISPR-Cas13b can be programmed to specifically target and degrade HBV RNAs to reduce HBV replication and protein expression, demonstrating its potential as a novel therapeutic option for chronic HBV infection. IMPACT AND IMPLICATIONS: There is an urgent need for new treatments that target multiple aspects of the HBV replication cycle. Here, we present CRISPR-Cas13b as a novel strategy to target HBV replication and protein expression paving the way for its development as a potential new treatment option for patients living with chronic hepatitis B.

Nanobody cocktails potently neutralize SARS-CoV-2 D614G N501Y variant and protect mice.

Neutralizing antibodies are important for immunity against SARS-CoV-2 and as therapeutics for the prevention and treatment of COVID-19. Here, we identified high-affinity nanobodies from alpacas immunized with coronavirus spike and receptor-binding domains (RBD) that disrupted RBD engagement with the human receptor angiotensin-converting enzyme 2 (ACE2) and potently neutralized SARS-CoV-2. Epitope mapping, X-ray crystallography, and cryo-electron microscopy revealed two distinct antigenic sites and showed two neutralizing nanobodies from different epitope classes bound simultaneously to the spike trimer. Nanobody-Fc fusions of the four most potent nanobodies blocked ACE2 engagement with RBD variants present in human populations and potently neutralized both wild-type SARS-CoV-2 and the N501Y D614G variant at concentrations as low as 0.1 nM. Prophylactic administration of either single nanobody-Fc or as mixtures reduced viral loads by up to 104-fold in mice infected with the N501Y D614G SARS-CoV-2 virus. These results suggest a role for nanobody-Fc fusions as prophylactic agents against SARS-CoV-2.

Correction: SARS-CoV-2 suppresses IFNß production mediated by NSP1, 5, 6, 15, ORF6 and ORF7b but does not suppress the effects of added interferon.

[This corrects the article DOI: 10.1371/journal.ppat.1009800.].

SARS-CoV-2 suppresses IFNß production mediated by NSP1, 5, 6, 15, ORF6 and ORF7b but does not suppress the effects of added interferon.

Type I Interferons (IFN-Is) are a family of cytokines which play a major role in inhibiting viral infection. Resultantly, many viruses have evolved mechanisms in which to evade the IFN-I response. Here we tested the impact of expression of 27 different SARS-CoV-2 genes in relation to their effect on IFN production and activity using three independent experimental methods. We identified six gene products; NSP6, ORF6, ORF7b, NSP1, NSP5 and NSP15, which strongly (>10-fold) blocked MAVS-induced (but not TRIF-induced) IFNß production. Expression of the first three of these SARS-CoV-2 genes specifically blocked MAVS-induced IFNß-promoter activity, whereas all six genes induced a collapse in IFNß mRNA levels, corresponding with suppressed IFNß protein secretion. Five of these six genes furthermore suppressed MAVS-induced activation of IFNλs, however with no effect on IFNα or IFNγ production. In sharp contrast, SARS-CoV-2 infected cells remained extremely sensitive to anti-viral activity exerted by added IFN-Is. None of the SARS-CoV-2 genes were able to block IFN-I signaling, as demonstrated by robust activation of Interferon Stimulated Genes (ISGs) by added interferon. This, despite the reduced levels of STAT1 and phospho-STAT1, was likely caused by broad translation inhibition mediated by NSP1. Finally, we found that a truncated ORF7b variant that has arisen from a mutant SARS-CoV-2 strain harboring a 382-nucleotide deletion associating with mild disease (Δ382 strain identified in Singapore & Taiwan in 2020) lost its ability to suppress type I and type III IFN production. In summary, our findings support a multi-gene process in which SARS-CoV-2 blocks IFN-production, with ORF7b as a major player, presumably facilitating evasion of host detection during early infection. However, SARS-CoV-2 fails to suppress IFN-I signaling thus providing an opportunity to exploit IFN-Is as potential therapeutic antiviral drugs.

A natural product compound inhibits coronaviral replication in vitro by binding to the conserved Nsp9 SARS-CoV-2 protein.

The Nsp9 replicase is a conserved coronaviral protein that acts as an essential accessory component of the multi-subunit viral replication/transcription complex. Nsp9 is the predominant substrate for the essential nucleotidylation activity of Nsp12. Compounds specifically interfering with this viral activity would facilitate its study. Using a native mass-spectrometry-based approach to screen a natural product library for Nsp9 binders, we identified an ent-kaurane natural product, oridonin, capable of binding to purified SARS-CoV-2 Nsp9 with micromolar affinities. By determining the crystal structure of the Nsp9-oridonin complex, we showed that oridonin binds through a conserved site near Nsp9's C-terminal GxxxG-helix. In enzymatic assays, oridonin's binding to Nsp9 reduces its potential to act as substrate for Nsp12's Nidovirus RdRp-Associated Nucleotidyl transferase (NiRAN) domain. We also showed using in vitro cellular assays oridonin, while cytotoxic at higher doses has broad antiviral activity, reducing viral titer following infection with either SARS-CoV-2 or, to a lesser extent, MERS-CoV. Accordingly, these preliminary findings suggest that the oridonin molecular scaffold may have the potential to be developed into an antiviral compound to inhibit the function of Nsp9 during coronaviral replication.

Enhancement of Antibody-Dependent Cellular Cytotoxicity and Phagocytosis in Anti-HIV-1 Human-Bovine Chimeric Broadly Neutralizing Antibodies.

No prophylactic vaccine has provided robust protection against human immunodeficiency virus type 1 (HIV-1). Vaccine-induced broadly neutralizing antibodies (bNAbs) have not been achieved in humans and most animals; however, cows vaccinated with HIV-1 envelope trimers produce bNAbs with unusually long third heavy complementarity-determining regions (CDRH3s). Alongside neutralization, Fc-mediated effector functions, including antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADP), may be critical for in vivo bNAb antiviral activity. Here, we aimed to augment the Fc-dependent effector functions of a chimeric human-bovine bNAb, NC-Cow1, which binds the CD4 binding site (CD4bs) and exhibits broader and more potent neutralization than most human CD4bs bNAbs by using an exceptionally long 60-amino acid (aa) CDRH3. The bovine variable region of NC-Cow1 was paired with a human IgG1 Fc region mutated to create the following three variants: G236R/L328R (GRLR) that abrogates Fc-gamma receptor (FcγR) binding, and two variants that enhance binding, namely, G236A/S239D/I332E (GASDIE) and G236A/S239D/A330L/I332E (GASDALIE). Both GASDIE and GASDALIE improved binding to human FcγRIIA and FcγRIIIA, enhanced human natural killer (NK) cell activation, and mediated higher levels of ADCC and ADP activity than the wild-type human IgG1 Fc. GASDALIE mediated higher phagocytic activity than GASDIE. As expected, GRLR eliminated binding to FcγRs and did not mediate ADCC or ADP. We demonstrated that mutations in the human Fc region of bovine chimeric antibodies with ultralong CDRH3s could enhance antibody effector functions while maintaining envelope binding and neutralization. This study will have significant implications in the development of multifunctional anti-HIV antibodies, which may be important to prevent HIV-1 transmission in an antibody-based topical microbicide. IMPORTANCE Despite successful antiviral chemotherapy, human immunodeficiency virus (HIV) is still a lifelong persistent virus, and no vaccine yet prevents HIV transmission. Topical microbicides offer an important alternative method to prevent sexual transmission of HIV-1. With the production of highly potent anti-HIV-1 broadly neutralizing antibodies (bNAbs) and multifunctional antibodies, monoclonal antibodies are now important prophylactic agents. Recently discovered anti-HIV-1 bovine bNAbs (with higher potency and breadth than most human bNAbs) could be novel candidates as potent topical microbicides. Our study is significant as it demonstrates the compatibility of combining bovine-derived neutralization with human-derived antibody-effector functions. This study is a new approach to antibody engineering that strengthens the feasibility of using high-potency bovine variable region bNAbs with augmented Fc function and promotes them as a strong candidate for antibody-mediated therapies.

Surface Inactivation of Highly Mutated SARS-CoV-2 Variants of Concern: Alpha, Delta, and Omicron.

Continued SARS-CoV-2 transmission among the human population has meant the evolution of the virus to produce variants of increased infectiousness and virulence, coined variants of concern (VOCs). The last wave of pandemic infections was driven predominantly by the delta VOC, but because of continued transmission and adaptive mutations, the more highly transmissible omicron variant emerged and is now dominant. However, due to waning immunity and emergence of new variants, vaccines alone cannot control the pandemic. The application of an antiviral coating to high-touch surfaces and physical barriers such as masks are an effective means to inactivate the virus and their spread. Here, we demonstrate an environmentally friendly water-borne polymer coating that can completely inactivate SARS-CoV-2 independent of the infectious variant. The polymer was designed to target the highly glycosylated spike protein on the virion surface and inactivate the virion by disruption of the viral membrane through a nano-mechanical process. Our findings show that, even with low amounts of coating on the surface (1 g/m2), inactivation of alpha, delta, and omicron VOCs and degradation of their viral genome were complete. Furthermore, our data shows that the polymer induces little to no skin sensitization in mice and is non-toxic upon oral ingestion in rats. We anticipate that our transparent polymer coating can be applied to face masks and many other surfaces to capture and inactivate the virus, aiding in the reduction of SARS-CoV-2 transmission and evolution of new variants of concern.

Tat IRES modulator of tat mRNA (TIM-TAM): a conserved RNA structure that controls Tat expression and acts as a switch for HIV productive and latent infection.

Tat protein is essential to fully activate HIV transcription and processing of viral mRNA, and therefore determines virus expression in productive replication and the establishment and maintenance of latent infection. Here, we used thermodynamic and structure analyses to define a highly conserved sequence-structure in tat mRNA that functions as Tat IRES modulator of tat mRNA (TIM-TAM). By impeding cap-dependent ribosome progression during authentic spliced tat mRNA translation, TIM-TAM stable structure impacts on timing and level of Tat protein hence controlling HIV production and infectivity along with promoting latency. TIM-TAM also adopts a conformation that mediates Tat internal ribosome entry site (IRES)-dependent translation during the early phases of infection before provirus integration. Our results document the critical role of TIM-TAM in Tat expression to facilitate virus reactivation from latency, with implications for HIV treatment and drug development.

Air-Liquid-Interface Differentiated Human Nose Epithelium: A Robust Primary Tissue Culture Model of SARS-CoV-2 Infection.

The global urgency to uncover medical countermeasures to combat the COVID-19 pandemic caused by the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has revealed an unmet need for robust tissue culture models that faithfully recapitulate key features of human tissues and disease. Infection of the nose is considered the dominant initial site for SARS-CoV-2 infection and models that replicate this entry portal offer the greatest potential for examining and demonstrating the effectiveness of countermeasures designed to prevent or manage this highly communicable disease. Here, we test an air-liquid-interface (ALI) differentiated human nasal epithelium (HNE) culture system as a model of authentic SARS-CoV-2 infection. Progenitor cells (basal cells) were isolated from nasal turbinate brushings, expanded under conditionally reprogrammed cell (CRC) culture conditions and differentiated at ALI. Differentiated cells were inoculated with different SARS-CoV-2 clinical isolates. Infectious virus release into apical washes was determined by TCID50, while infected cells were visualized by immunofluorescence and confocal microscopy. We demonstrate robust, reproducible SARS-CoV-2 infection of ALI-HNE established from different donors. Viral entry and release occurred from the apical surface, and infection was primarily observed in ciliated cells. In contrast to the ancestral clinical isolate, the Delta variant caused considerable cell damage. Successful establishment of ALI-HNE is donor dependent. ALI-HNE recapitulate key features of human SARS-CoV-2 infection of the nose and can serve as a pre-clinical model without the need for invasive collection of human respiratory tissue samples.

Optimal preparation of SARS-CoV-2 viral transport medium for culture.

INTRODUCTION: The sudden arrival of the COVID-19 pandemic placed significant stresses on supply chains including viral transport medium (VTM). The VTM that was urgently required needed to support viral replication, as well as other routine diagnostic approaches. We describe the preparation and validation testing of VTM for rapidly expanding diagnostic testing, where the capacity of the VTM to preserve viral integrity, for culture, isolation and full sequence analysis, was maintained. METHODS: VTM was prepared using different methods of sterilization then 'spiked' with virus. The VTM was investigated using viral culture in Vero cells, and for nucleic acid detection by quantitative PCR. RESULTS: The best results were obtained by filter and autoclave-based sterilization. The VTM proved robust for culture-based analyses provided the inoculated VTM was stored at 4 °C, and tested within 48 h. The filtered VTM also supported PCR-based diagnosis for at least 5 days when the mock inoculated VTM was held at room temperature. DISCUSSION: The manual handling of VTM production, including filling and sterilization, was optimized. SARS-CoV-2 was spiked into VTM to assess different sterilization methods and measure the effects of storage time and temperature upon VTM performance. While most diagnostic protocols will not require replication competent virus, the use of high quality VTM will allow for the next phase of laboratory analysis in the COVID-19 pandemic, including drug and antibody susceptibility analysis of re-isolated SARS-CoV-2, and for the testing of vaccine escape mutants.

Evaluation of Serological Tests for SARS-CoV-2: Implications for Serology Testing in a Low-Prevalence Setting.

BACKGROUND: Robust serological assays are essential for long-term control of the COVID-19 pandemic. Many recently released point-of-care (PoCT) serological assays have been distributed with little premarket validation. METHODS: Performance characteristics for 5 PoCT lateral flow devices approved for use in Australia were compared to a commercial enzyme immunoassay (ELISA) and a recently described novel surrogate virus neutralization test (sVNT). RESULTS: Sensitivities for PoCT ranged from 51.8% (95% confidence interval [CI], 43.1%-60.4%) to 67.9% (95% CI, 59.4%-75.6%), and specificities from 95.6% (95% CI, 89.2%-98.8%) to 100.0% (95% CI, 96.1%-100.0%). ELISA sensitivity for IgA or IgG detection was 67.9% (95% CI, 59.4%-75.6%), increasing to 93.8% (95% CI, 85.0%-98.3%) for samples >14 days post symptom onset. sVNT sensitivity was 60.9% (95% CI, 53.2%-68.4%), rising to 91.2% (95% CI, 81.8%-96.7%) for samples >14 days post symptom onset, with specificity 94.4% (95% CI, 89.2%-97.5%). CONCLUSIONS: Performance characteristics for COVID-19 serological assays were generally lower than those reported by manufacturers. Timing of specimen collection relative to onset of illness or infection is crucial in reporting of performance characteristics for COVID-19 serological assays. The optimal algorithm for implementing serological testing for COVID-19 remains to be determined, particularly in low-prevalence settings.

Infrared Based Saliva Screening Test for COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in an unprecedented need for diagnostic testing that is critical in controlling the spread of COVID-19. We propose a portable infrared spectrometer with purpose-built transflection accessory for rapid point-of-care detection of COVID-19 markers in saliva. Initially, purified virion particles were characterized with Raman spectroscopy, synchrotron infrared (IR) and AFM-IR. A data set comprising 171 transflection infrared spectra from 29 subjects testing positive for SARS-CoV-2 by RT-qPCR and 28 testing negative, was modeled using Monte Carlo Double Cross Validation with 50 randomized test and model sets. The testing sensitivity was 93 % (27/29) with a specificity of 82 % (23/28) that included positive samples on the limit of detection for RT-qPCR. Herein, we demonstrate a proof-of-concept high throughput infrared COVID-19 test that is rapid, inexpensive, portable and utilizes sample self-collection thus minimizing the risk to healthcare workers and ideally suited to mass screening.

Functional properties and sequence variation of HTLV-1 p13.

Human T cell leukemia virus type-1 (HTLV-1) was the first retrovirus found to cause cancer in humans, but the mechanisms that drive the development of leukemia and other diseases associated with HTLV-1 infection remain to be fully understood. This review describes the functional properties of p13, an 87-amino acid protein coded by HTLV-1 open reading frame II (orf-II). p13 is mainly localized in the inner membrane of the mitochondria, where it induces potassium (K+) influx and reactive oxygen species (ROS) production, which can trigger either proliferation or apoptosis, depending on the ROS setpoint of the cell. Recent evidence indicates that p13 may influence the cell's innate immune response to viral infection and the infected cell phenotype. Association of the HTLV-1 transcriptional activator, Tax, with p13 increases p13's stability, leads to its partial co-localization with Tax in nuclear speckles, and reduces the ability of Tax to interact with the transcription cofactor CBP/p300. Comparison of p13 sequences isolated from HTLV-1-infected individuals revealed a small number of amino acid variations in the domains controlling the subcellular localization of the protein. Disruptive mutations of p13 were found in samples obtained from asymptomatic patients with low proviral load. p13 sequences of HTLV-1 subtype C isolates from indigenous Australian patients showed a high degree of identity among each other, with all samples containing a pattern of 5 amino acids that distinguished them from other subtypes. Further characterization of p13's functional properties and sequence variants may lead to a deeper understanding of the impact of p13 as a contributor to the clinical manifestations of HTLV-1 infection.

Impact of Hepatitis B Virus Coinfection on Human T-Lymphotropic Virus Type 1 Clonality in an Indigenous Population of Central Australia.

The prevalence of human T-cell lymphotropic virus type 1 (HTLV-1) and hepatitis B virus (HBV) coinfection is high in certain Indigenous Australian populations, but its impact on HTLV-1 has not been described. We compared 2 groups of Indigenous adults infected with HTLV-1, either alone or coinfected with HBV. The 2 groups had a similar HTLV-1 proviral load, but there was a significant increase in clonal expansion of HTLV-1-infected lymphocytes in coinfected asymptomatic individuals. The degree of clonal expansion was correlated with the titer of HBV surface antigen. We conclude that HTLV-1/HBV coinfection may predispose to HTLV-1-associated malignant disease.

Role of HTLV-1 orf-I encoded proteins in viral transmission and persistence.

The human T cell leukemia virus type 1 (HTVL-1), first reported in 1980 by Robert Gallo's group, is the etiologic agent of both cancer and inflammatory diseases. Despite approximately 40 years of investigation, the prognosis for afflicted patients remains poor with no effective treatments. The virus persists in the infected host by evading the host immune response and inducing proliferation of infected CD4+ T-cells. Here, we will review the role that viral orf-I protein products play in altering intracellular signaling, protein expression and cell-cell communication in order to escape immune recognition and promote T-cell proliferation. We will also review studies of orf-I mutations found in infected patients and their potential impact on viral load, transmission and persistence. Finally, we will compare the orf-I gene in HTLV-1 subtypes as well as related STLV-1.

p30 protein: a critical regulator of HTLV-1 viral latency and host immunity.

The extraordinarily high prevalence of HTLV-1 subtype C (HTLV-1C) in some isolated indigenous communities in Oceania and the severity of the health conditions associated with the virus impress the great need for basic and translational research to prevent and treat HTLV-1 infection. The genome of the virus's most common subtype, HTLV-1A, encodes structural, enzymatic, and regulatory proteins that contribute to viral persistence and pathogenesis. Among these is the p30 protein encoded by the doubly spliced Tax-orf II mRNA, a nuclear/nucleolar protein with both transcriptional and post-transcriptional activity. The p30 protein inhibits the productive replication cycle via nuclear retention of the mRNA that encodes for both the viral transcriptional trans-activator Tax, and the Rex proteins that regulate the transport of incompletely spliced viral mRNA to the cytoplasm. In myeloid cells, p30 inhibits the PU-1 transcription factor that regulates interferon expression and is a critical mediator of innate and adaptive immunity. Furthermore, p30 alters gene expression, cell cycle progression, and DNA damage responses in T-cells, raising the hypothesis that p30 may directly contribute to T cell transformation. By fine-tuning viral expression while also inhibiting host innate responses, p30 is likely essential for viral infection and persistence. This concept is supported by the finding that macaques, a natural host for the closely genetically related simian T-cell leukemia virus 1 (STLV-1), exposed to an HTLV-1 knockout for p30 expression by a single point mutation do not became infected unless reversion and selection of the wild type HTLV-1 genotype occurs. All together, these data suggest that inhibition of p30 may help to curb and eventually eradicate viral infection by exposing infected cells to an effective host immune response.

Multiplex Droplet Digital PCR Assay for Quantification of Human T-Cell Leukemia Virus Type 1 Subtype c DNA Proviral Load and T Cells from Blood and Respiratory Exudates Sampled in a Remote Setting.

During human T-cell leukemia virus type 1 (HTLV-1) infection, the frequency of cells harboring an integrated copy of viral cDNA, the proviral load (PVL), is the main risk factor for progression of HTLV-1-associated diseases. Accurate quantification of provirus by droplet digital PCR (ddPCR) is a powerful diagnostic tool with emerging uses for monitoring viral expression. Current ddPCR techniques quantify HTLV-1 PVL in terms of whole genomic cellular material, while the main targets of HTLV-1 infection are CD4+ and CD8+ T cells. Our understanding of HTLV-1 proliferation and the amount of viral burden present in different compartments is limited. Recently a sensitive ddPCR assay was applied to quantifying T cells by measuring loss of germ line T-cell receptor genes as method of distinguishing non-T-cell from recombined T-cell DNA. In this study, we demonstrated and validated novel applications of the duplex ddPCR assay to quantify T cells from various sources of human genomic DNA (gDNA) extracted from frozen material (peripheral blood mononuclear cells [PBMCs], bronchoalveolar lavage fluid, and induced sputum) from a cohort of remote Indigenous Australians and then compared the T-cell measurements by ddPCR to the prevailing standard method of flow cytometry. The HTLV-1 subtype c (HTLV-1c) PVL was then calculated in terms of extracted T-cell gDNA from various compartments. Because HTLV-1c preferentially infects CD4+ T cells, and the amount of viral burden correlates with HTLV-1c disease pathogenesis, application of this ddPCR assay to accurately measure HTLV-1c-infected T cells can be of greater importance for clinical diagnostics and prognostics as well as monitoring therapeutic applications.

Identification of Native and Posttranslationally Modified HLA-B*57:01-Restricted HIV Envelope Derived Epitopes Using Immunoproteomics.

The recognition of pathogen-derived peptides by T lymphocytes is the cornerstone of adaptive immunity, whereby intracellular antigens are degraded in the cytosol and short peptides assemble with class I human leukocyte antigen (HLA) molecules in the ER. These peptide-HLA complexes egress to the cell surface and are scrutinized by cytotoxic CD8+ T-cells leading to the eradication of the infected cell. Here, naturally presented HLA-B*57:01 bound peptides derived from the envelope protein of the human immunodeficiency virus (HIVenv) are identified. HIVenv peptides are present at a very small percentage of the overall HLA-B*57:01 peptidome (<0.1%) and both native and posttranslationally modified forms of two distinct HIV peptides are identified. Notably, a peptide bearing a natively encoded C-terminal tryptophan residue is also present in a modified form containing a kynurenine residue. Kynurenine is a major product of tryptophan catabolism and is abundant during inflammation and infection. Binding of these peptides at a molecular level and their immunogenicity in preliminary functional studies are examined. Modest immune responses are observed to the modified HIVenv peptide, highlighting a potential role for kynurenine-modified peptides in the immune response to HIV and other viral infections.