Murine cytomegalovirus downregulates ERAAP and induces an unconventional T cell response to self.

The endoplasmic reticulum aminopeptidase associated with antigen processing (ERAAP) plays a crucial role in shaping the peptide-major histocompatibility complex (MHC) class I repertoire and maintaining immune surveillance. While murine cytomegalovirus (MCMV) has multiple strategies for manipulating the antigen processing pathway to evade immune responses, the host has also developed ways to counter viral immune evasion. In this study, we find that MCMV modulates ERAAP and induces an interferon γ (IFN-γ)-producing CD8+ T cell effector response that targets uninfected ERAAP-deficient cells. We observe that ERAAP downregulation during infection leads to the presentation of the self-peptide FL9 on non-classical Qa-1b, thereby eliciting Qa-1b-restricted QFL T cells to proliferate in the liver and spleen of infected mice. QFL T cells upregulate effector markers upon MCMV infection and are sufficient to reduce viral load after transfer to immunodeficient mice. Our study highlights the consequences of ERAAP dysfunction during viral infection and provides potential targets for anti-viral therapies.

SARS-CoV-2 Envelope-mediated Golgi pH dysregulation interferes with ERAAP retention in cells.

Endoplasmic reticulum (ER) aminopeptidase associated with antigen processing (ERAAP) trims peptide precursors in the ER for presentation by major histocompatibility (MHC)-I molecules to surveying CD8+ T-cells. This function allows ERAAP to regulate the nature and quality of the peptide repertoire and, accordingly, the resulting immune responses. We recently showed that infection with murine cytomegalovirus leads to a dramatic loss of ERAAP levels in infected cells. In mice, this loss is associated with the activation of QFL T-cells, a subset of T-cells that monitor ERAAP integrity and eliminate cells experiencing ERAAP dysfunction. In this study, we aimed to identify host factors that regulate ERAAP expression level and determine whether these could be manipulated during viral infections. We performed a CRISPR knockout screen and identified ERp44 as a factor promoting ERAAP retention in the ER. ERp44's interaction with ERAAP is dependent on the pH gradient between the ER and Golgi. We hypothesized that viruses that disrupt the pH of the secretory pathway interfere with ERAAP retention. Here, we demonstrate that expression of the Envelope (E) protein from Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) leads to Golgi pH neutralization and consequently decrease of ERAAP intracellular levels. Furthermore, SARS-CoV-2-induced ERAAP loss correlates with its release into the extracellular environment. ERAAP's reliance on ERp44 and a functioning ER/Golgi pH gradient for proper localization and function led us to propose that ERAAP serves as a sensor of disturbances in the secretory pathway during infection and disease.

Detecting Intact Virus Using Exogenous Oligonucleotide Labels.

The COVID-19 pandemic has revealed how an emerging pathogen can cause a sudden and dramatic increase in demand for viral testing. Testing pooled samples could meet this demand; however, the sensitivity of reverse transcription quantitative polymerase chain reaction (RT-qPCR), the gold standard, significantly decreases with an increasing number of samples pooled. Here, we introduce detection of intact virus by exogenous-nucleotide reaction (DIVER), a method that quantifies intact virus and is robust to sample dilution. As demonstrated using two models of severe acute respiratory syndrome coronavirus 2, DIVER first tags membraned particles with exogenous oligonucleotides, then captures the tagged particles on beads functionalized with a virus-specific capture agent (in this instance, angiotensin-converting enzyme 2), and finally quantifies the oligonucleotide tags using qPCR. Using spike-presenting liposomes and spike-pseudotyped lentivirus, we show that DIVER can detect 1 × 105 liposomes and 100 plaque-forming units of lentivirus and can successfully identify positive samples in pooling experiments. Overall, DIVER is well positioned for efficient sample pooling and clinical validation.

Toward Community Surveillance: Detecting Intact SARS-CoV-2 Using Exogeneous Oligonucleotide Labels.

The persistence of the COVID-19 pandemic demands a dramatic increase in testing efficiency. Testing pooled samples for SARS-CoV-2 could meet this need; however, the sensitivity of RT-qPCR, the gold standard, significantly decreases with an increasing number of samples pooled. Here, we introduce DIVER, a method that quantifies intact virus and is robust to sample dilution. DIVER first tags viral particles with exogeneous oligonucleotides, then captures the tagged particles on ACE2-functionalized beads, and finally quantifies the oligonucleotide tags using qPCR. Using spike-presenting liposomes and Spike-pseudotyped lentivirus as SARS-CoV-2 models, we show that DIVER can detect 1×10 5 liposomes and 100 pfu lentivirus and can successfully identify positive samples in pooling experiments. Overall, DIVER is well-positioned for efficient sample pooling and expanded community surveillance.

An Unusual MHC Molecule Generates Protective CD8+ T Cell Responses to Chronic Infection.

The CD8+ T cell response to the intracellular parasite Toxoplasma gondii varies dramatically between mouse strains, resulting in stark differences in control of the parasite. Protection in BALB/c mice can be attributed to an unusually strong and protective MHC-1 Ld-restricted CD8+ T cell response directed against a peptide derived from the parasite antigen GRA6. The MHC-1 Ld molecule has limited peptide binding compared to conventional MHC molecules such as Kb or Db, which correlates with polymorphisms associated with "elite control" of HIV in humans. To investigate the link between the unusual MHC-1 molecule Ld and the generation of "elite controller" CD8+ T cell responses, we compared the GRA6-Ld specific T cell response to the well-studied OVA-Kb specific response, and demonstrated that GRA6-Ld specific T cells are significantly more protective and resistant to exhaustion in chronic T. gondii infection. To further investigate the connection between limited peptide presentation and robust T cell responses, we used CRISPR/Cas9 to generate mice with a point mutation (W97R) in the peptide-binding groove of Ld that results in broader peptide binding. We investigated the effect of this Ld W97R mutation on another robust Ld-restricted response against the IE1 peptide during Murine Cytomegalovirus (MCMV) infection. This mutation leads to an increase in exhaustion markers in the IE1-Ld specific CD8+ T cell response. Our results indicate that limited peptide binding by MHC-1 Ld correlates with the development of robust and protective CD8+ T cell responses that may avoid exhaustion during chronic infection.

Epitope Mapping and Fine Specificity of Human T and B Cell Responses for Novel Candidate Blood-Stage Malaria Vaccine P27A.

P27A is a novel synthetic malaria vaccine candidate derived from the blood stage Plasmodium falciparum protein Trophozoite Exported Protein 1 (TEX1/PFF0165c). In phase 1a/1b clinical trials in malaria unexposed adults in Switzerland and in malaria pre-exposed adults in Tanzania, P27A formulated with Alhydrogel and GLA-SE adjuvants induced antigen-specific antibodies and T-cell activity. The GLA-SE adjuvant induced significantly stronger humoral responses than the Alhydrogel adjuvant. Groups of pre-exposed and unexposed subjects received identical vaccine formulations, which supported the comparison of the cellular and humoral response to P27A in terms of fine specificity and affinity for populations and adjuvants. Globally, fine specificity of the T and B cell responses exhibited preferred recognized sequences and did not highlight major differences between adjuvants or populations. Affinity of anti-P27A antibodies was around 10-8 M in all groups. Pre-exposed volunteers presented anti-P27A with higher affinity than unexposed volunteers. Increasing the dose of GLA-SE from 2.5 to 5 µg in pre-exposed volunteers improved anti-P27A affinity and decreased the number of recognized epitopes. These results indicate a higher maturation of the humoral response in pre-exposed volunteers, particularly when immunized with P27A formulated with 5 µg GLA-SE.

The Candidate Blood-stage Malaria Vaccine P27A Induces a Robust Humoral Response in a Fast Track to the Field Phase 1 Trial in Exposed and Nonexposed Volunteers.

Background: P27A is an unstructured 104mer synthetic peptide from Plasmodium falciparum trophozoite exported protein 1 (TEX1), the target of human antibodies inhibiting parasite growth. The present project aimed at evaluating the safety and immunogenicity of P27A peptide vaccine in malaria-nonexposed European and malaria-exposed African adults. Methods: This study was designed as a staggered, fast-track, randomized, antigen and adjuvant dose-finding, multicenter phase 1a/1b trial, conducted in Switzerland and Tanzania. P27A antigen (10 or 50 µg), adjuvanted with Alhydrogel or glucopyranosil lipid adjuvant stable emulsion (GLA-SE; 2.5 or 5 µg), or control rabies vaccine (Verorab) were administered intramuscularly to 16 malaria-nonexposed and 40 malaria-exposed subjects on days 0, 28, and 56. Local and systemic adverse events (AEs) as well as humoral and cellular immune responses were assessed after each injection and during the 34-week follow-up. Results: Most AEs were mild to moderate and resolved completely within 48 hours. Systemic AEs were more frequent in the formulation with alum as compared to GLA-SE, whereas local AEs were more frequent after GLA-SE. No serious AEs occurred. Supported by a mixed Th1/Th2 cell-mediated immunity, P27A induced a marked specific antibody response able to recognize TEX1 in infected erythrocytes and to inhibit parasite growth through an antibody-dependent cellular inhibition mechanism. Incidence of AEs and antibody responses were significantly lower in malaria-exposed Tanzanian subjects than in nonexposed European subjects. Conclusions: The candidate vaccine P27A was safe and induced a particularly robust immunogenic response in combination with GLA-SE. This formulation should be considered for future efficacy trials. Clinical Trials Registration: NCT01949909, PACTR201310000683408.

An Mtb-Human Protein-Protein Interaction Map Identifies a Switch between Host Antiviral and Antibacterial Responses.

Although macrophages are armed with potent antibacterial functions, Mycobacterium tuberculosis (Mtb) replicates inside these innate immune cells. Determinants of macrophage intrinsic bacterial control, and the Mtb strategies to overcome them, are poorly understood. To further study these processes, we used an affinity tag purification mass spectrometry (AP-MS) approach to identify 187 Mtb-human protein-protein interactions (PPIs) involving 34 secreted Mtb proteins. This interaction map revealed two factors involved in Mtb pathogenesis-the secreted Mtb protein, LpqN, and its binding partner, the human ubiquitin ligase CBL. We discovered that an lpqN Mtb mutant is attenuated in macrophages, but growth is restored when CBL is removed. Conversely, Cbl-/- macrophages are resistant to viral infection, indicating that CBL regulates cell-intrinsic polarization between antibacterial and antiviral immunity. Collectively, these findings illustrate the utility of this Mtb-human PPI map for developing a deeper understanding of the intricate interactions between Mtb and its host.