The HLA-II immunopeptidome of SARS-CoV-2 (preprint)

Targeted synthetic vaccines have the potential to transform our response to viral outbreaks; yet the design of these vaccines requires a comprehensive knowledge of viral immunogens, including T-cell epitopes. Having previously mapped the SARS-CoV-2 HLA-I landscape, here we report viral peptides that are naturally processed and loaded onto HLA-II complexes in infected cells. We identified over 500 unique viral peptides from canonical proteins, as well as from overlapping internal open reading frames (ORFs), revealing, for the first time, the contribution of internal ORFs to the HLA-II peptide repertoire. Most HLA-II peptides co-localized with the known CD4+ T cell epitopes in COVID-19 patients. We also observed that two reported immunodominant regions in the SARS-CoV-2 membrane protein are formed at the level of HLA-II presentation. Overall, our analyses show that HLA-I and HLA-II pathways target distinct viral proteins, with the structural proteins accounting for most of the HLA-II peptidome and non-structural and non-canonical proteins accounting for the majority of the HLA-I peptidome. These findings highlight the need for a vaccine design that incorporates multiple viral elements harboring CD4+ and CD8+ T cell epitopes to maximize the vaccine effectiveness.

Vaccine serologic responses among transplant patients associate with COVID-19 infection and T peripheral helper cells (preprint)

Background: Therapeutically immunosuppressed transplant recipients exhibit attenuated responses to COVID-19 vaccines. To better understand the immune alterations that determined poor vaccine response, we correlated quantities of circulating T and B cell subsets at baseline with longitudinal serologic responses to SARS-CoV-2 mRNA vaccination in heart and lung transplant recipients. Methods: Samples at baseline and at approximately 8 and 30 days after each vaccine dose for 22 heart and lung transplant recipients with no history of COVID-19, four heart and lung transplant recipients with prior COVID-19 infection, and 12 healthy controls undergoing vaccination were analyzed. Anti-spike protein receptor binding domain (RBD) IgG and pseudovirus neutralization activity were measured. Proportions of B and T cell subsets at baseline were comprehensively quantitated. Results: At 8-30 days post vaccination, healthy controls displayed robust anti-RBD IgG responses, whereas heart and lung transplant recipients showed minimally increased responses. A parallel absence of activity was observed in pseudovirus neutralization. In contrast, three of four (75%) transplant recipients with prior COVID-19 infection displayed robust responses at levels comparable to controls. Baseline levels of activated PD-1+ HLA-DR+ CXCR5- CD4+ T cells (also known as T peripheral helper [TPH] cells) and CD4+ T cells strongly predicted the ability to mount a response. Conclusions: Immunosuppressed patients have defective vaccine responses but can be induced to generate neutralizing antibodies after SARS-CoV-2 infection. Strong correlations of vaccine responsiveness with baseline TPH and CD4+ T cell numbers highlights a role for T helper activity in B cell differentiation into antibody secreting cells during vaccine response.

SARS-CoV-2 Viremia is Associated with Distinct Proteomic Pathways and Predicts COVID-19 Outcomes (preprint)

BackgroundSevere Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) plasma viremia has been associated with severe disease and death in coronavirus disease 2019 (COVID-19) in small-scale cohort studies. The mechanisms behind this association remain elusive. MethodsWe evaluated the relationship between SARS-CoV-2 viremia, disease outcome, inflammatory and proteomic profiles in a cohort of COVID-19 emergency department participants. SARS-CoV-2 viral load was measured using qRT-PCR based platform. Proteomic data were generated with Proximity Extension Assay (PEA) using the Olink platform. ResultsThree hundred participants with nucleic acid test-confirmed COVID-19 were included in this study. Levels of plasma SARS-CoV-2 viremia at the time of presentation predicted adverse disease outcomes, with an adjusted odds ratio (aOR) of 10.6 (95% confidence interval [CI] 4.4, 25.5, P<0.001) for severe disease (mechanical ventilation and/or 28-day mortality) and aOR of 3.9 (95%CI 1.5, 10.1, P=0.006) for 28-day mortality. Proteomic analyses revealed prominent proteomic pathways associated with SARS-CoV-2 viremia, including upregulation of SARS-CoV-2 entry factors (ACE2, CTSL, FURIN), heightened markers of tissue damage to the lungs, gastrointestinal tract, endothelium/vasculature and alterations in coagulation pathways. ConclusionsThese results highlight the cascade of vascular and tissue damage associated with SARS-CoV-2 plasma viremia that underlies its ability to predict COVID-19 disease outcomes.

Plasma proteomics reveals tissue-specific cell death and mediators of cell-cell interactions in severe COVID-19 patients (preprint)

COVID-19 has caused over 1 million deaths globally, yet the cellular mechanisms underlying severe disease remain poorly understood. By analyzing several thousand plasma proteins in 306 COVID-19 patients and 78 symptomatic controls over serial timepoints using two complementary approaches, we uncover COVID-19 host immune and non-immune proteins not previously linked to this disease. Integration of plasma proteomics with nine published scRNAseq datasets shows that SARS-CoV-2 infection upregulates monocyte/macrophage, plasmablast, and T cell effector proteins. By comparing patients who died to severely ill patients who survived, we identify dynamic immunomodulatory and tissue-associated proteins associated with survival, providing insights into which host responses are beneficial and which are detrimental to survival. We identify intracellular death signatures from specific tissues and cell types, and by associating these with angiotensin converting enzyme 2 (ACE2) expression, we map tissue damage associated with severe disease and propose which damage results from direct viral infection rather than from indirect effects of illness. We find that disease severity in lung tissue is driven by myeloid cell phenotypes and cell-cell interactions with lung epithelial cells and T cells. Based on these results, we propose a model of immune and epithelial cell interactions that drive cell-type specific and tissue-specific damage in severe COVID-19.

A cell-free antibody engineering platform rapidly generates SARS-CoV-2 neutralizing antibodies (preprint)

Antibody engineering technologies face increasing demands for speed, reliability and scale. We developed CeVICA, a cell-free antibody engineering platform that integrates a novel generation method and design for camelid heavy-chain antibody VHH domain-based synthetic libraries, optimized in vitro selection based on ribosome display and a computational pipeline for binder prediction based on CDR-directed clustering. We applied CeVICA to engineer antibodies against the Receptor Binding Domain (RBD) of the SARS-CoV-2 spike proteins and identified >800 predicted binder families. Among 14 experimentally-tested binders, 6 showed inhibition of pseudotyped virus infection. Antibody affinity maturation further increased binding affinity and potency of inhibition. Additionally, the unique capability of CeVICA for efficient and comprehensive binder prediction allowed retrospective validation of the fitness of our synthetic VHH library design and revealed direction for future refinement. CeVICA offers an integrated solution to rapid generation of divergent synthetic antibodies with tunable affinities in vitro and may serve as the basis for automated and highly parallel antibody generation.

SARS-CoV-2 infected cells present HLA-I peptides from canonical and out-of-frame ORFs (preprint)

T cell-mediated immunity may play a critical role in controlling and establishing protective immunity against SARS-CoV-2 infection; yet the repertoire of viral epitopes responsible for T cell response activation remains mostly unknown. Identification of viral peptides presented on class I human leukocyte antigen (HLA-I) can reveal epitopes for recognition by cytotoxic T cells and potential incorporation into vaccines. Here, we report the first HLA-I immunopeptidome of SARS-CoV-2 in two human cell lines at different times post-infection using mass spectrometry. We found HLA-I peptides derived not only from canonical ORFs, but also from internal out-of-frame ORFs in Spike and Nucleoprotein not captured by current vaccines. Proteomics analyses of infected cells revealed that SARS-CoV-2 may interfere with antigen processing and immune signaling pathways. Based on the endogenously processed and presented viral peptides that we identified, we estimate that a pool of 24 peptides would provide one or more peptides for presentation by at least one HLA allele in 99% of the human population. These biological insights and the list of naturally presented SARS-CoV-2 peptides will facilitate data-driven selection of peptides for immune monitoring and vaccine development.

Safe and effective two-in-one replicon-and-VLP minispike vaccine for COVID-19 (preprint)

The large SARS-CoV-2 spike (S) protein is the main target of current COVID-19 vaccine candidates but can induce non-neutralizing antibodies, which may cause vaccination-induced complications or enhancement of COVID-19 disease. Besides, encoding of a functional S in replication-competent virus vector vaccines may result in the emergence of viruses with altered or expanded tropism. Here, we have developed a safe single round rhabdovirus replicon vaccine platform for enhanced presentation of the S receptor-binding domain (RBD). Structure-guided design was employed to build a chimeric minispike comprising the globular RBD linked to a transmembrane stem-anchor sequence derived from rabies virus (RABV) glycoprotein (G). Vesicular stomatitis virus (VSV) and RABV replicons encoding the minispike not only allowed expression of the antigen at the cell surface but also incorporation into the envelope of secreted non-infectious particles, thus combining classic vector-driven antigen expression and particulate virus-like particle (VLP) presentation. A single dose of a prototype replicon vaccine, VSV{Delta}G-minispike-eGFP (G), stimulated high titers of SARS-CoV-2 neutralizing antibodies in mice, equivalent to those found in COVID-19 patients. Boost immunization with the identical replicon further enhanced neutralizing activity. These results demonstrate that rhabdovirus minispike replicons represent effective and safe alternatives to vaccination approaches using replication-competent viruses and/or the entire S antigen.

Transmission of SARS-COV-2 from China to Europe and West Africa: a detailed phylogenetic analysis. (preprint)

Background: SARS-CoV-2, the virus causing the Covid-19 pandemic emerged in December 2019 in China and raised fears that it could overwhelm healthcare systems worldwide. In June 2020, all African countries registered human infections with SARS-CoV-2. The virus is mutating steadily and this is monitored by a well curated database of viral nucleotide sequences from samples taken from infected individual thus enabling phylogenetic analysis and phenotypic associations. Methods: We downloaded from the GISAID database, SARS-CoV-2 sequences established from four West African countries Ghana, Gambia, Senegal and Nigeria and then performed phylogenetic analysis employing the nextstrain pipeline. Based on mutations found within the sequences we calculated and visualized statistics characterizing clades according to the GISAID nomenclature. Results: We found country-specific patterns of viral clades: the later Europe-associated G-clades predominantly in Senegal and Gambia, and combinations of the earlier (L, S, V) and later clades in Ghana and Nigeria. Contrary to our expectations, the later Europe-associated G-clades emerged before the earlier clades. Detailed analysis of distinct samples showed that some of the earlier clades might have circulated latently and some reflect migration routes via Mali and Tunisia. Conclusions: The distinct patterns of viral clades in the West African countries point at its emergence from Europe and China via Asia and Europe. The observation that the later clades emerged before the earlier clades could be simply due to founder effects or due to latent circulation of the earlier clades. Only a marginal correlation of the G-clades associated with the D614G mutation could be identified with the relatively low case fatality (0.6-3.2).