Vaccines: Reaching for higher branches after the low hanging fruit has been picked

Vaccines represent one of the most significant advances by medicine in addressing infectious diseases being responsible for the elimination of a few and the virtual eradication of several other scourges. In addition, medicine is also beginning to appreciate the potential applicability of vaccines beyond infectious diseases to areas such as oncology. During any emerging infectious disease public health event, availability of a vaccine figures prominently in the assessment of the capability to address the threat most effectively. A more rapid deployment of new vaccines would enable more effective management of emerging threats. Efforts to streamline manufacture and production of vaccines have yielded some success. Vaccination enhancements such as temperature stability, adjuvants, and alternative delivery devices are needed for improving overall effectiveness. At the same time, animal model testing of candidate vaccines have displayed variable success in predicting immunogenicity and efficacy in humans. Examples of efforts in these latter arenas will be discussed to highlight the potential of newer technologies and approaches that may further improve the capability to bring vaccines forward towards clinical evaluation in a timelier manner to impact responses to emerging infectious diseases

A Novel Epstein-Barr Virus Glycoprotein gp150 Expressed from the BDLF3 Open Reading Frame

AbstractAnalysis of cDNAs mapping to the BamHI D fragment of the Epstein-Barr virus (EBV) genome indicates that the BDLF3 open reading frame, which is predicted to encode a type 1 membrane protein of 234 amino acids, is expressed as an unspliced message. Expression of the open reading frame as a recombinant protein in vaccinia virus reveals a glycoprotein that has both N- and O-linked sugars. Antibodies made to the recombinant protein immunoprecipitate a late glycoprotein with a mobility of approximately 150,000 Da from EBV-producing cells. The glycoprotein is associated with the virion. Antibodies to it appear to react primarily with carbohydrate and do not demonstrate neutralizing activity

Multiple HLA-All-restricted cytotoxic T-lymphocyte epitopes of different immunogenicities in the EpsteinBarr virus-encoded nuclear antigen 4

Epstein-Barr virus (EBV), a ubiquitous herpesvirus, induces potent HLA class I-restricted cytotoxic T-lymphocyte (CTL) responses. Analyses of target antigen choice have shown that the very strong CTL responses which are often observed through the HLA All allele map are due almost entirely to a single transformation-associated EBV protein, the nuclear antigen EBNA4. Here, we sought to determine the number and relative immunogenicities of HLA All-restricted epitopes within this 938-amino-acid protein. An initial screening with a series of recombinant vaccinia virus vectors encoding progressively truncated forms of EBNA4 was followed by peptide sensitization experiments using overlapping 14-or 15-mers from the entire sequence. These two approaches allowed the identification of five epitope regions located between residues 101 and 115, 416 and 429, 396 and 410, 481 and 495, and 551 and 564 of the EBNA4 molecule. CTL preparations from all seven HLA All-positive donors tested had demonstrable reactivities against the 416-to-429 peptide, whereas reactivities against the other epitopes either tended to be lost on serial passage or, for some of the donors, were never detected. The immunodominance of the 416-to-429 epitope was further supported by peptide dilution assays using polyclonal effectors and by CTL cloning experiments. Analysis of the 416-to-429 region identified the nanomer 416-424 (IVTDFSVIK) as the cognate peptide. This peptide was able to sensitize targets to lysis by All-restricted CTL clones at concentrations as low as 5 x 10-14 M. Epstein-Barr virus (EBV) is a widespread lymphotropic herpesvirus which causes infectious mononucleosis and is strongly linked to at least three lymphoid malignancies: endemic Burkitt's lymphoma, immunoblastic B-cell lymphomas of immunosuppressed patients, and a subset of Hodgkin's lymphomas (7-9). Primary EBV infection of immunocompetent hosts is usually asymptomatic and leads to the establishment of a life-long carrier state, whereby the virus persists within the B-cell compartment of healthy carriers (5). These infected B lymphocytes can proliferate in vitro, giving rise to lymphoblastoid cell lines (LCLs) which express at least eight latency-associated viral antigens: the nuclear antigens EBNA1 to -6 and the membrane proteins LMP1 and -2 (reviewed in reference 13). The recent demonstration that the immunoblastic lymphomas occurring in immunosuppressed individuals represent the in vivo outgrowth of EBV-positive LCL-like cells (6) emphasizes the role of immune surveillance in controlling this potentially lymphomagenic virus. EBV induces long-lasting cytotoxic T-lymphocyte (CTL) memory in the infected host. Thus, EBV-specific CTL precursors can be reactivated in relatively large numbers from the T-cell pool of EBV-seropositive donors by challenging in vitro with autologous virus-infected B cell

High Frequency of Cytomegalovirus-Specific Cytotoxic T-Effector Cells in HLA-A*0201-Positive Subjects during Multiple Viral Coinfections

How the cellular immune response copes with diverse antigenic competition is poorly understood. Responses of virus-specific cytotoxic T lymphocytes (CTL) were examined longitudinally in an individual coinfected with human immunodeficiency virus type 1 (HIV-1), Epstein-Barr virus (EBV), and cytomegalovirus (CMV). CTL responses to all 3 viruses were quantified by limiting dilution analysis and staining with HLA-A*0201 tetrameric complexes folded with HIV-1, EBV, and CMV peptides. A predominance of CMV-pp65-speciflc CTL was found, with a much lower frequency of CTL to HIV-1 Gag and Pol and to EBV-BMLF1 and LMP2. The high frequency of CMV-speciflc CTL, compared with HIV-1- and EBV-specific CTL, was confirmed in an additional 16 HLA-A*0201-positive virus-coinfected subjects. Therefore, the human immune system can mount CTL responses to multiple viral antigens simultaneously, albeit with different strength

The National COVID Cohort Collaborative (N3C): Rationale, design, infrastructure, and deployment.

OBJECTIVE: Coronavirus disease 2019 (COVID-19) poses societal challenges that require expeditious data and knowledge sharing. Though organizational clinical data are abundant, these are largely inaccessible to outside researchers. Statistical, machine learning, and causal analyses are most successful with large-scale data beyond what is available in any given organization. Here, we introduce the National COVID Cohort Collaborative (N3C), an open science community focused on analyzing patient-level data from many centers. MATERIALS AND METHODS: The Clinical and Translational Science Award Program and scientific community created N3C to overcome technical, regulatory, policy, and governance barriers to sharing and harmonizing individual-level clinical data. We developed solutions to extract, aggregate, and harmonize data across organizations and data models, and created a secure data enclave to enable efficient, transparent, and reproducible collaborative analytics. RESULTS: Organized in inclusive workstreams, we created legal agreements and governance for organizations and researchers; data extraction scripts to identify and ingest positive, negative, and possible COVID-19 cases; a data quality assurance and harmonization pipeline to create a single harmonized dataset; population of the secure data enclave with data, machine learning, and statistical analytics tools; dissemination mechanisms; and a synthetic data pilot to democratize data access. CONCLUSIONS: The N3C has demonstrated that a multisite collaborative learning health network can overcome barriers to rapidly build a scalable infrastructure incorporating multiorganizational clinical data for COVID-19 analytics. We expect this effort to save lives by enabling rapid collaboration among clinicians, researchers, and data scientists to identify treatments and specialized care and thereby reduce the immediate and long-term impacts of COVID-19

Increased Incidence of Vestibular Disorders in Patients With SARS-CoV-2

OBJECTIVE: Determine the incidence of vestibular disorders in patients with SARS-CoV-2 compared to the control population. STUDY DESIGN: Retrospective. SETTING: Clinical data in the National COVID Cohort Collaborative database (N3C). METHODS: Deidentified patient data from the National COVID Cohort Collaborative database (N3C) were queried based on variant peak prevalence (untyped, alpha, delta, omicron 21K, and omicron 23A) from covariants.org to retrospectively analyze the incidence of vestibular disorders in patients with SARS-CoV-2 compared to control population, consisting of patients without documented evidence of COVID infection during the same period. RESULTS: Patients testing positive for COVID-19 were significantly more likely to have a vestibular disorder compared to the control population. Compared to control patients, the odds ratio of vestibular disorders was significantly elevated in patients with untyped (odds ratio [OR], 2.39; confidence intervals [CI], 2.29-2.50; CONCLUSIONS: The incidence of vestibular disorders differed between COVID-19 variants and was significantly elevated in COVID-19-positive patients compared to the control population. These findings have implications for patient counseling and further research is needed to discern the long-term effects of these findings

Six RNA Viruses and Forty-One Hosts: Viral Small RNAs and Modulation of Small RNA Repertoires in Vertebrate and Invertebrate Systems

We have used multiplexed high-throughput sequencing to characterize changes in small RNA populations that occur during viral infection in animal cells. Small RNA-based mechanisms such as RNA interference (RNAi) have been shown in plant and invertebrate systems to play a key role in host responses to viral infection. Although homologs of the key RNAi effector pathways are present in mammalian cells, and can launch an RNAi-mediated degradation of experimentally targeted mRNAs, any role for such responses in mammalian host-virus interactions remains to be characterized. Six different viruses were examined in 41 experimentally susceptible and resistant host systems. We identified virus-derived small RNAs (vsRNAs) from all six viruses, with total abundance varying from “vanishingly rare” (less than 0.1% of cellular small RNA) to highly abundant (comparable to abundant micro-RNAs “miRNAs”). In addition to the appearance of vsRNAs during infection, we saw a number of specific changes in host miRNA profiles. For several infection models investigated in more detail, the RNAi and Interferon pathways modulated the abundance of vsRNAs. We also found evidence for populations of vsRNAs that exist as duplexed siRNAs with zero to three nucleotide 3′ overhangs. Using populations of cells carrying a Hepatitis C replicon, we observed strand-selective loading of siRNAs onto Argonaute complexes. These experiments define vsRNAs as one possible component of the interplay between animal viruses and their hosts

Transient Selection During Vaccinia Virus Recombination with Insertion Vectors Without Selectable Markers

Isolation of recombinant viruses after DNA transfection remains the most timeconsuming and labor-intensive feature in the production of vaccinia virus (VV) recombinants. This procedure has been aided by the design and incorporation of both positive and negative selectable markers into the insertion vectors. Use of these selectable markers is not always feasible or desirable. In the absence of selectable markers, individual plaque characterization is required. A method of transient selection during transfection and recombination is described that does not require that any selectable marker be present in the insertion vector sequence. This method relies on the co-transfection of a guanine phosphoribosyl transferase (gpt) selectable marker on an unlinked plasmid into VV-infected cells pretreated for gpt selection. Following recombination, the output virus can be plated on cells without any further selection and screened for recombinant virus. While the total recombinant virus population is slightly reduced, the total progeny virus is reduced even further, resulting in more than sevenfold enhancement in the frequency of recombinant virus, which allows for more efficient identification