564. SARS-CoV-2 Ferritin Nanoparticle Vaccines Elicit Broad SARS Coronavirus Immunogenicity

Background The zoonotic emergence of SARS-CoV-2 quickly developed into a global pandemic. Multiple vaccine platforms have been advanced to clinical trials and emergency use authorization. The recent emergence of SARS-CoV-2 virus variants with Spike receptor-binding domain (RBD) and N-terminal domain (NTD) mutations, highlights the need for next-generation vaccines that can elicit immune responses that are resilient against Spike mutations. Methods Using a structure-based vaccine design approach, we developed multiple optimized SARS-CoV-2 nanoparticle immunogens that recapitulate the structural and antigenic profile of the SARS-CoV-2 prefusion spike. We assessed these immunogens in murine immunogenicity studies and in a K18-hACE2 transgenic mouse model with a SARS-CoV-2 challenge. Immune sera from vaccinated mice were assessed for SARS-CoV-2 binding, and neutralization against SARS-CoV-2, variants of concern, and the heterologous SARS-CoV-1 virus. Results In combination with a liposomal-saponin based adjuvant (ALFQ), these immunogens induced robust binding, ACE2-inhibition, and authentic virus and pseudovirus neutralization. A Spike-Ferritin nanoparticle (SpFN) vaccine elicited neutralizing ID50 titers >10,000 after a single immunization, while RBD-Ferritin (RFN) nanoparticle immunogens elicited ID50 titer values >10,000 values after two immunizations. Purified antibody from SpFN- or RFN-immunized mice was transfused into K18-ACE2 transgenic mice and challenged with a high-dose SARS-CoV-2 virus stock. In order to understand the breadth of vaccine-elicited antibody responses, we analyzed SpFN- and RBD-FN-immunized animal sera against a set of heterologous SARS-CoV-2 RBD variants and SARS-CoV RBD. High binding titers with ACE2-blocking activity were observed against SARS-CoV-2 variants and the heterologous SARS-CoV-1 RBD. Furthermore, both SpFN- and RFN-immunized animal sera showed SARS-CoV-1 neutralizing ID50 titers of >2000. Conclusion These observations highlight the importance of SARS-CoV-2 neutralizing antibody levels in providing protection against emerging SARS-like coronaviruses and provide a robust platform for pandemic preparedness. Structure-based design enables development of a SARS-CoV-2 nanoparticle immunogen. Disclosures All Authors: No reported disclosures

Cross-neutralization of SARS-CoV-2 by HIV-1 specific broadly neutralizing antibodies and polyclonal plasma

Cross-reactive epitopes (CREs) are similar epitopes on viruses that are recognized or neutralized by same antibodies. The S protein of SARS-CoV-2, similar to type I fusion proteins of viruses such as HIV-1 envelope (Env) and influenza hemagglutinin, is heavily glycosylated. Viral Env glycans, though host derived, are distinctly processed and thereby recognized or accommodated during antibody responses. In recent years, highly potent and/or broadly neutralizing human monoclonal antibodies (bnAbs) that are generated in chronic HIV-1 infections have been defined. These bnAbs exhibit atypical features such as extensive somatic hypermutations, long complementary determining region (CDR) lengths, tyrosine sulfation and presence of insertions/deletions, enabling them to effectively neutralize diverse HIV-1 viruses despite extensive variations within the core epitopes they recognize. As some of the HIV-1 bnAbs have evolved to recognize the dense viral glycans and cross-reactive epitopes (CREs), we assessed if these bnAbs cross-react with SARS-CoV-2. Several HIV-1 bnAbs showed cross-reactivity with SARS-CoV-2 while one HIV-1 CD4 binding site bnAb, N6, neutralized SARS-CoV-2. Furthermore, neutralizing plasma antibodies of chronically HIV-1 infected children showed cross neutralizing activity against SARS-CoV-2 pseudoviruses. Collectively, our observations suggest that human monoclonal antibodies tolerating extensive epitope variability can be leveraged to neutralize pathogens with related antigenic profile.

Challenge of Interpreting Cytoplasmic CD20 Stain in Diffuse Large B Cell Lymphoma Biopsy Specimen of Patients with Steroid Usage

Lymphoma work-up involves immunohistochemical stains to help reach the diagnosis. It is imperative to have clinical information and sound knowledge of staining pattern of antibodies to avoid misinterpretation of results. We describe two cases in which pre-biopsy steroid hindered antigenic profile, leading to “cytoplasmic granular staining” and causing delay.

Trichuris trichiura egg extract proteome reveals potential diagnostic targets and immunomodulators

Embryonated eggs are the infectious developmental stage of Trichuris trichiura and are the primary stimulus for the immune system of the definitive host. The intestinal-dwelling T. trichiura affects an estimated 465 million people worldwide with an estimated global burden of disease of 640 000 DALYs (Disability Adjusted Life Years). In Latin America and the Caribbean, trichuriasis is the most prevalent soil transmitted helminthiasis in the region (12.3%; 95% CI). The adverse health consequences impair childhood school performance and reduce school attendance resulting in lower future wage-earning capacity. The accumulation of the long-term effects translates into poverty promoting sequelae and a cycle of impoverishment. Each infective T. trichiura egg carries the antigens needed to face the immune system with a wide variety of proteins present in the shell, larvae’s surface, and the accompanying fluid that contains their excretions/secretions. We used a proteomic approach with tandem mass spectrometry to investigate the proteome of soluble non-embryonated egg extracts of T. trichiura obtained from naturally infected African green monkeys (Chlorocebus sabaeus). A total of 231 proteins were identified, 168 of them with known molecular functions. The proteome revealed common proteins families which are known to play roles in energy and metabolism; the cytoskeleton, muscle and motility; proteolysis; signaling; the stress response and detoxification; transcription and translation; and lipid binding and transport. In addition to the study of the T. trichiura non-embryonated egg proteome, the antigenic profile of the T. trichiura non-embryonated egg and female soluble proteins against serum antibodies from C. sabaeus naturally infected with trichuriasis was investigated. We used an immunoproteomic approach by Western blot and tandem mass spectrometry from the corresponding SDS-PAGE gels. Vitellogenin N and VWD and DUF1943 domain containing protein, poly-cysteine and histidine tailed protein isoform 2, heat shock protein 70, glyceraldehyde-3-phosphate dehydrogenase, actin, and enolase, were among the potential immunoactive proteins. To our knowledge, this is the first study on the T. trichiura non-embryonated egg proteome as a novel source of information on potential targets for immunodiagnostics and immunomodulators from a neglected tropical disease. This initial list of T. trichiura non-embryonated egg proteins (proteome and antigenic profile) can be used in future research on the immunobiology and pathogenesis of human trichuriasis and the treatment of human intestinal immune-related diseases.

Hemagglutination Inhibition (HAI) antibody landscapes after vaccination with H7Nx virus like particles

BackgroundA systemic evaluation of the antigenic differences of the H7 influenza hemagglutinin (HA) proteins, especially for the viruses isolated after 2016, are limited. The purpose of this study was to investigate the antigenic differences of major H7 strains with an ultimate aim to discover H7 HA proteins that can elicit protective receptor-binding antibodies against co-circulating H7 influenza strains.MethodA panel of eight H7 influenza strains were selected from 3,633 H7 HA amino acid sequences identified over the past two decades (2000–2018). The sequences were expressed on the surface of virus like particles (VLPs) and used to vaccinate C57BL/6 mice. Serum samples were collected and tested for hemagglutination-inhibition (HAI) activity. The vaccinated mice were challenged with lethal dose of H7N9 virus, A/Anhui/1/2013.ResultsVLPs expressing the H7 HA antigens elicited broadly reactive antibodies each of the selected H7 HAs, except the A/Turkey/Italy/589/2000 (Italy/00) H7 HA. A putative glycosylation due to an A169T substitution in antigenic site B was identified as a unique antigenic profile of Italy/00. Introduction of the putative glycosylation site (H7 HA-A169T) significantly altered the antigenic profile of HA of the A/Anhui/1/2013 (H7N9) strain.ConclusionThis study identified key amino acid mutations that result in severe vaccine mismatches for future H7 epidemics. Future universal influenza vaccine candidates will need to focus on viral variants with these key mutations.

Hemagglutination Inhibition (HAI) Antibody Landscapes after Vaccination with diverse H7 hemagglutinin (HA) proteins

BackgroundA systemic evaluation of the antigenic differences of the H7 influenza hemagglutinin (HA) proteins, especially for the viruses isolated after 2016, are limited. The purpose of this study was to investigate the antigenic differences of major H7 strains with an ultimate aim to discover H7 HA proteins that can elicit protective receptor-blocking antibodies against co-circulating H7 influenza strains.MethodA panel of nine H7 influenza strains were selected from 3,633 H7 HA amino acid sequences identified over the past two decades (2000-2018). The sequences were expressed on the surface of virus like particles (VLPs) and used to vaccinate C57BL/6 mice. Serum samples were collected and tested for hemagglutination-inhibition (HAI) activity. The vaccinated mice were challenged with lethal dose of H7N9 virus, A/Anhui/1/2013.ResultsVLPs expressing the H7 HA antigens elicited broadly reactive antibodies each of the selected H7 HAs, except the A/Turkey/Italy/589/2000 (Italy/00) H7 HA. A putative glycosylation due to an A169T substitution in antigenic site B was identified as a unique antigenic profile of Italy/00. Introduction of the putative glycosylation site (H7 HA-A169T) significantly altered the antigenic profile of HA of the A/Anhui/1/2013 (H7N9) strain.ConclusionThis study identified key amino acid mutations that result in severe vaccine mismatches for future H7 epidemics. Future universal influenza vaccine candidates will need to focus on viral variants with these key mutations.

Elucidating tumor immunosurveillance and immunoediting: a comprehensive review

The action of the immune system against neoplastic diseases has become one of the main sources of research. The biological pathways of this system are known to contribute in limiting the progression and elimination of the tumor, and are delineated by concepts and mechanisms of immunosurveillance and immunoediting. Immunosurveillance is considered the process by which the immune system recognizes and inhibits the neoplastic process. The concept of immunoediting arises in the sense that immune system is able to shape the antigenic profile of the tumor due to selective pressure, based on the stages of tumor elimination, balance and evasion. The immune response occurs against tumor antigens and changes in the tumor microenvironment, involving different components of the innate immune system, such as T cells, natural Killer cells, B lymphocytes and macrophages. In this sense, knowing these concepts and understanding their respective mechanisms becomes essential in the investigation of new strategies for cancer prevention and cure. Thus, this review presents historical aspects and definitions of immunosurveillance and tumor immunoediting, with emphasis on its importance and applicability, such as on the different methods used in immunotherapy.

Proteomic Analysis Of Trichuris Trichiura Egg Extract Reveals Potential Immunomodulators And Diagnostic Targets

Background: Trichuris trichiura embryonated eggs are the infectious developmental stage and the first signal to the immune system of the definitive host. Each infective T. trichiura egg carries the antigens needed to challenge the immune system with a wide variety of proteins present in the shell, larvae’s surface, and the accompanying fluid that contains their excretions/secretions. The parasite eggs constitute the first antigenic stimuli to evoke the host response to this intestinal parasite with direct life cycle and enteric development.Methods: The soluble egg extract of T. trichiura obtained from naturally infected African green monkeys (Chlorocebus sabaeus) was investigated using a proteomic approach by mass spectrometry. The antigenic profile of the egg soluble proteins against sera IgG from C. sabaeus with trichuriasis was also investigated by Western blot and LC-MS/MS from the corresponding SDS-PAGE gel.Results: A total of 231 proteins were accurately identified, 168 with known molecular functions. The proteome of the egg lysate revealed common protein families including energy and metabolism; cytoskeleton, motility and muscle; proteolysis; signaling; stress and detoxification; transcription and translation and; lipid binding and transport. Vitellogenin N and VWD and DUF1943 domain containing protein, Poly-cysteine and histidine tailed protein isoform 2, Heat shock protein 70, Glyceraldehyde-3-phosphate dehydrogenase, Actin and Enolase, were among the potential immunoactive proteins. Conclusions: To our knowledge, this study represents the first attempt to identify the proteome of the T. trichiura egg extract as a novel source of immunomodulators and targets for immunodiagnosis able to contribute to the treatment of human autoimmune diseases and to the control of this neglected disease.

Genomic and serologic characterization of enterovirus A71 brainstem encephalitis

ObjectiveIn 2016, Catalonia experienced a pediatric brainstem encephalitis outbreak caused by enterovirus A71 (EV-A71). Conventional testing identified EV in the periphery but rarely in CSF. Metagenomic next-generation sequencing (mNGS) and CSF pan-viral serology (VirScan) were deployed to enhance viral detection and characterization.MethodsRNA was extracted from the CSF (n = 20), plasma (n = 9), stool (n = 15), and nasopharyngeal samples (n = 16) from 10 children with brainstem encephalitis and 10 children with meningitis or encephalitis. Pathogens were identified using mNGS. Available CSF from cases (n = 12) and pediatric other neurologic disease controls (n = 54) were analyzed with VirScan with a subset (n = 9 and n = 50) validated by ELISA.ResultsmNGS detected EV in all samples positive by quantitative reverse transcription polymerase chain reaction (qRT-PCR) (n = 25). In qRT-PCR-negative samples (n = 35), mNGS found virus in 23% (n = 8, 3 CSF samples). Overall, mNGS enhanced EV detection from 42% (25/60) to 57% (33/60) (p-value = 0.013). VirScan and ELISA increased detection to 92% (11/12) compared with 46% (4/12) for CSF mNGS and qRT-PCR (p-value = 0.023). Phylogenetic analysis confirmed the EV-A71 strain clustered with a neurovirulent German EV-A71. A single amino acid substitution (S241P) in the EVA71 VP1 protein was exclusive to the CNS in one subject.ConclusionmNGS with VirScan significantly increased the CNS detection of EVs relative to qRT-PCR, and the latter generated an antigenic profile of the acute EV-A71 immune response. Genomic analysis confirmed the close relation of the outbreak EV-A71 and neuroinvasive German EV-A71. A S241P substitution in VP1 was found exclusively in the CSF.

Fibronectin and Its Role in Human Infective Diseases

Fibronectin is a multidomain glycoprotein ubiquitously detected in extracellular fluids and matrices of a variety of animal and human tissues where it functions as a key link between matrices and cells. Fibronectin has also emerged as the target for a large number of microorganisms, particularly bacteria. There are clear indications that the binding of microorganism’ receptors to fibronectin promotes attachment to and infection of host cells. Each bacterium may use different receptors which recognize specific fibronectin domains, mostly the N-terminal domain and the central cell-binding domain. In many cases, fibronectin receptors have actions over and above that of simple adhesion: In fact, adhesion is often the prerequisite for invasion and internalization of microorganisms in the cells of colonized tissues. This review updates the current understanding of fibronectin receptors of several microorganisms with emphasis on their biochemical and structural properties and the role they can play in the onset and progression of host infection diseases. Furthermore, we describe the antigenic profile and discuss the possibility of designing adhesion inhibitors based on the structure of the fibronectin-binding site in the receptor or the receptor-binding site in fibronectin.