A Synthetic Peptide CTL Vaccine Targeting Nucleocapsid Confers Protection from SARS-CoV-2 Challenge in Rhesus Macaques.

BACKGROUND: Persistent transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has given rise to a COVID-19 pandemic. Several vaccines, conceived in 2020, that evoke protective spike antibody responses are being deployed in mass public health vaccination programs. Recent data suggests, however, that as sequence variation in the spike genome accumulates, some vaccines may lose efficacy. METHODS: Using a macaque model of SARS-CoV-2 infection, we tested the efficacy of a peptide-based vaccine targeting MHC class I epitopes on the SARS-CoV-2 nucleocapsid protein. We administered biodegradable microspheres with synthetic peptides and adjuvants to rhesus macaques. Unvaccinated control and vaccinated macaques were challenged with 1 × 108 TCID50 units of SARS-CoV-2, followed by assessment of clinical symptoms and viral load, chest radiographs, and sampling of peripheral blood and bronchoalveolar lavage (BAL) fluid for downstream analysis. RESULTS: Vaccinated animals were free of pneumonia-like infiltrates characteristic of SARS-CoV-2 infection and presented with lower viral loads relative to controls. Gene expression in cells collected from BAL samples of vaccinated macaques revealed a unique signature associated with enhanced development of adaptive immune responses relative to control macaques. CONCLUSIONS: We demonstrate that a room temperature stable peptide vaccine based on known immunogenic HLA class I bound CTL epitopes from the nucleocapsid protein can provide protection against SARS-CoV-2 infection in nonhuman primates.

Paired SARS-CoV-2 spike protein mutations observed during ongoing SARS-CoV-2 viral transfer from humans to minks and back to humans.

A mutation analysis of SARS-CoV-2 genomes collected around the world sorted by sequence, date, geographic location, and species has revealed a large number of variants from the initial reference sequence in Wuhan. This analysis also reveals that humans infected with SARS-CoV-2 have infected mink populations in the Netherlands, Denmark, United States, and Canada. In these animals, a small set of mutations in the spike protein receptor binding domain (RBD), often occurring in specific combinations, has transferred back into humans. The viral genomic mutations in minks observed in the Netherlands and Denmark show the potential for new mutations on the SARS-CoV-2 spike protein RBD to be introduced into humans by zoonotic transfer. Our data suggests that close attention to viral transfer from humans to farm animals and pets will be required to prevent build-up of a viral reservoir for potential future zoonotic transfer.

An effective CTL peptide vaccine for Ebola Zaire Based on Survivors' CD8+ targeting of a particular nucleocapsid protein epitope with potential implications for COVID-19 vaccine design.

The 2013-2016 West Africa EBOV epidemic was the biggest EBOV outbreak to date. An analysis of virus-specific CD8+ T-cell immunity in 30 survivors showed that 26 of those individuals had a CD8+ response to at least one EBOV protein. The dominant response (25/26 subjects) was specific to the EBOV nucleocapsid protein (NP). It has been suggested that epitopes on the EBOV NP could form an important part of an effective T-cell vaccine for Ebola Zaire. We show that a 9-amino-acid peptide NP44-52 (YQVNNLEEI) located in a conserved region of EBOV NP provides protection against morbidity and mortality after mouse adapted EBOV challenge. A single vaccination in a C57BL/6 mouse using an adjuvanted microsphere peptide vaccine formulation containing NP44-52 is enough to confer immunity in mice. Our work suggests that a peptide vaccine based on CD8+ T-cell immunity in EBOV survivors is conceptually sound and feasible. Nucleocapsid proteins within SARS-CoV-2 contain multiple Class I epitopes with predicted HLA restrictions consistent with broad population coverage. A similar approach to a CTL vaccine design may be possible for that virus.

Eliciting cytotoxic T-lymphocyte responses from synthetic vectors containing one or two epitopes in a C57BL/6 mouse model using peptide-containing biodegradable microspheres and adjuvants.

We describe a vaccine delivery mechanism consisting of a synthetic, non-living vector of large d,l poly(lactic-co-glycolic) acid (PLGA) microspheres that carry specific cytotoxic T lymphocyte (CTL) epitopes. We demonstrate in mice that it can be used to elicit substantial interferon gamma ELISPOT responses to more than one specific epitope in the same individual. Our data suggest that a superior adjuvant configuration for the formulation is to place a TLR-9 agonist CpG inside the microsphere and a TLR-4 agonist MPLA in the injectate solution. This finding contrasts with the observations of others. Our approach provides a means to elicit immune responses efficiently to select epitopes, which may be important for an effective vaccine against HIV.

Double-stranded RNA-dependent protein kinase (PKR) is a stress-responsive kinase that induces NFkappaB-mediated resistance against mercury cytotoxicity.

The interferon-inducible, double-stranded (ds)RNA-dependent protein kinase (PKR) plays a major role in antiviral defense mechanisms where it down-regulates translation via phosphorylation of eukaryotic translation initiation factor 2alpha. PKR is also involved in the activation of nuclear factor kappaB (NFkappaB) through activation of the IkappaB kinase complex. Activation of PKR can occur in the absence of dsRNA and in such case is controlled by intracellular regulators like the PKR-activating protein (PACT), the PKR inhibitor p58(IPK), or heat-shock proteins (Hsp). These regulators are activated by stress stimuli, supporting a role for PKR in response to stress; however the final outcome of PKR activation in stress situations is unclear. We present here evidence that expression and activation of PKR contributes to an increased cellular resistance to mercury cytotoxicity. In two cell lines constitutively expressing PKR (THP-1 and Molt-3), treatment with the PKR inhibitor 2-aminopurine increases their sensitivity to mercury. In contrast, Ramos cells, which do not constitutively express PKR, present an increased resistance to mercury when PKR expression is induced by polyIC or interferon-beta treatment. This protective effect is inhibited by 2-aminopurine. We also show that exposure of Ramos cells to mercury leads to the induction of Hsp70. Treatment of cells with Hsp70 or NFkappaB inhibitors suppresses the PKR-dependent protection. We propose a model where PKR, modulated by Hsp70, activates a NFkappaB-mediated protective pathway. Because the cytotoxicity of mercury is primarily due to the generation of reactive oxygen species, our results suggest a more general function of PKR in the mechanisms of cellular response to oxidative stress.

2',5'-Oligoadenylate size is critical to protect RNase L against proteolytic cleavage in chronic fatigue syndrome.

A dysregulation in the 2',5'-oligoadenylate (2-5A)-dependent RNase L antiviral pathway has been detected in peripheral blood mononuclear cells (PBMC) of chronic fatigue syndrome (CFS) patients, which is characterized by upregulated 2-5A synthetase and RNase L activities, as well as by the presence of a low molecular weight (LMW) 2-5A-binding protein of 37-kDa related to RNase L. This truncated protein has been shown to originate from proteolytic cleavage of the native 83-kDa RNase L by m-calpain and human leukocyte elastase (HLE). We investigated the possible role of 2-5A oligomers in the proteolytic action toward the endonuclease and show that incubation of CFS PBMC extracts with 2-5A trimer and tetramer, but not with the dimer, results in a significant protection of the native 83-kDa RNase L against cleavage by endogenous and purified proteases. Similar results are obtained with a purified recombinant RNase L. An analysis of the size of 2-5A oligomers produced by the catalytic activity of the 2-5A synthetase present in PBMC extracts further shows that samples containing the 37-kDa RNase L preferentially produce 2-5A dimers instead of higher oligomers. Taken together, our results indicate that homodimerization of RNase L by 2-5A oligomers higher than the dimer prevents its cleavage by proteolytic enzymes. The presence of the truncated 37-kDa RNase L in PBMC extracts is therefore likely to result, not only from the abnormal activation of inflammatory proteases, but also from a dysregulation in 2-5A synthetase induction or activation towards the preferential production of 2-5A dimers.

Type I interferons induce proteins susceptible to act as thyroid receptor (TR) corepressors and to signal the TR for destruction by the proteasome: possible etiology for unexplained chronic fatigue.

In some patients complaining of chronic fatigue such as those suffering from the chronic fatigue syndrome (CFS), no underlying physical cause can be clearly identified and they typically present a normal thyroid function. Several studies indicate a dysregulation in the type I interferons (IFN-alpha/beta) pathway in CFS resulting in a sustained upregulation of 2('),5(')-oligoadenylate synthetases (2-5OAS). Likewise, patients treated with IFN-alpha/beta usually complain of severe fatigue as a limiting side effect. Beside the 2-5OAS, IFN-alpha/beta induce also the expression of three closely related proteins of unknown function termed the 2-5OAS-like (2-5OASL) proteins. The amino acid sequences of the 2-5OASL proteins display 96% identity with the partial sequence of the thyroid receptor interacting protein (TRIP) 14, further contain two typical thyroid hormone receptor (TR) coregulator domains and feature two ubiquitin C-terminal domains. From these observations, we raise the hypothesis that the 2-5OASL proteins are TRIPs capable of, respectively, repressing TR transactivation and/or signaling the receptor for destruction by the proteasome. Such molecular mechanisms could explain the development of a clinical hypothyroid state in presence of a normal thyroid function.

Structural and functional features of the 37-kDa 2-5A-dependent RNase L in chronic fatigue syndrome.

A 2',5'-oligoadenylate (2-5A)-dependent 37-kDa form of RNase L has been reported in extracts of peripheral blood mononuclear cells (PBMC) from individuals with chronic fatigue syndrome (CFS). In the current study, analytic gel permeation FPLC, azido photoaffinity labeling, two-dimensional (2-D) gel electrophoresis, and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) have been used to examine the biochemical relationship between the 80-kDa RNase L in healthy control PBMC and the 37-kDa RNase L in PBMC from individuals with CFS. Like the 80-kDa RNase L, the 37-kDa RNase L is present as a catalytically inactive heterodimer complex with the RNase L inhibitor (RLI). Formation of a 37-kDa RNase L-RLI complex indicates that the 37-kDa RNase L is structurally similar to the 80-kDa RNase L at the N-terminus, which contains the 2-5A binding domain. The enzymatically active monomer form of 37-kDa RNase L resolved by 2-D gel electrophoresis has a pI of 6.1. RT-PCR and Southern blot analyses demonstrated that the 37-kDa RNase L is not formed by alternative splicing. In-gel tryptic digestion of the 37-kDa RNase L that was excised from 2-D gels and subsequent MALDI-MS analysis identified three peptide masses that are identical to three predicted peptide masses in the 80-kDa RNase L. The electrophoretic mobility of 2-5A azido photolabeled/immunoprecipitated 37-kDa RNase L was the same under reducing and nonreducing conditions. The results presented show that the 37-kDa form of RNase L in PBMC shares structural and functional features with the native 80-kDa RNase L, in particular in the 2-5A binding and catalytic domains.

Biochemical evidence for a novel low molecular weight 2-5A-dependent RNase L in chronic fatigue syndrome.

Previous studies from this laboratory have demonstrated a statistically significant dysregulation in several key components of the 2',5'-oligoadenylate (2-5A) synthetase/RNase L and PKR antiviral pathways in chronic fatigue syndrome (CFS) (Suhadolnik et al. Clin Infect Dis 18, S96-104, 1994; Suhadolnik et al. In Vivo 8, 599-604, 1994). Two methodologies have been developed to further examine the upregulated RNase L activity in CFS. First, photoaffinity labeling of extracts of peripheral blood mononuclear cells (PBMC) with the azido 2-5A photoaffinity probe, [32P]pApAp(8-azidoA), followed by immunoprecipitation with a polyclonal antibody against recombinant, human 80-kDa RNase L and analysis under denaturing conditions. A subset of individuals with CFS was identified with only one 2-5A binding protein at 37 kDa, whereas in extracts of PBMC from a second subset of CFS PBMC and from healthy controls, photolabeled/immunoreactive 2-5A binding proteins were detected at 80, 42, and 37 kDa. Second, analytic gel permeation HPLC was completed under native conditions. Extracts of healthy control PBMC revealed 2-5A binding and 2-5A-dependent RNase L enzyme activity at 80 and 42 kDa as determined by hydrolysis of poly(U)-3'-[32P]pCp. A subset of CFS PBMC contained 2-5A binding proteins with 2-5A-dependent RNase L enzyme activity at 80, 42, and 30 kDa. However, a second subset of CFS PBMC contained 2-5A binding and 2-5A-dependent RNase L enzyme activity only at 30 kDa. Evidence is provided indicating that the RNase L enzyme dysfunction in CFS is more complex than previously reported.

Prolonged N-myc protein half-life in a neuroblastoma cell line lacking N-myc amplification.

Genomic amplification of the oncogene N-myc is associated with rapid tumor progression and poor prognosis in patients with neuroblastoma (NB). However, 40% of NBs which lack N-myc amplification are also clinically aggressive. Factors other than N-myc copy number must therefore play a role in determining tumor progression in these NBs. We have established an unusual human NB cell line (NBL-S) from the primary tumor of a patient with rapidly progressive disease which lacks N-myc amplification. The doubling time in vitro (48 h) and the time from injection of 2 x 10(7) cells to detectable tumors in nude mice (46 days) in similar to NB cell lines with amplified N-myc. However, karyotype analysis reveals no evidence of double minutes (DMs), homogeneously staining regions (HSRs), or chromosome 1p deletions, features commonly seen in NB cell lines. The cells have the cell surface phenotype typical of N-myc amplified NB (HLA-A,B,C negative and HSAN 1.2 positive), and similar to other NB cell lines, N-myc RNA and protein are expressed. Interestingly, the half-life of the N-myc protein in NBL-S is prolonged (approximately 100 min) compared to the short N-myc protein half-life previously described in N-myc amplified NB cell lines (approximately 30 min). Because N-myc protein is thought to have a regulatory role, prolongation of the half-life of this protein may be an important factor in the regulation of growth in NBs which lack N-myc amplification and rapidly progress.

Characterization of a novel myeloma cell line, MM.1.

A myeloma cell line, MM.1, has been established from the peripheral blood cells of a patient with immunoglobulin A myeloma. MM.1 grows in suspension either singly or in small clusters and secretes lambda-light chain. Phenotypically, MM.1 cells lack most B cell antigens, but they do express human leukocyte antigen DR, PCA-1, and T9 and T10 antigens. Molecular analysis of MM.1 demonstrates that it is negative for the presence of the Epstein-Barr virus genome. Southern analysis of MM.1 detected a rearrangement of the lambda-light chain gene, and Northern analysis revealed high levels of lambda gene expression. Cytogenetic analysis of the MM.1 cell line revealed the presence of seven related chromosomally abnormal cell lines characterized by numerical and structural aberrations, and it revealed five nonclonal abnormal cells. The most notable abnormality is a reciprocal translocation involving band q24.3 of chromosome 12 and band q32.3 of chromosome 14; translocations involving 14q32 are frequently observed in neoplasms of B cell origin.

Single copies of the N-myc oncogene in neuroblastomas from children presenting with the syndrome of opsoclonus-myoclonus.

Patients with neuroblastoma who present with the syndrome of opsoclonus and myoclonus enjoy a remarkably good prognosis independent of their stage of disease or their age at diagnosis. The presence of N-myc amplification also has been found to be an independent prognostic factor in neuroblastoma. Patients with multicopy N-myc tumors have rapid tumor progression whereas those with single-copy tumors have a significantly better progression-free survival. The authors examined four primary, untreated neuroblastomas for the N-myc copy number from patients who presented with opsoclonus and myoclonus. All four tumors had single copies of N-myc, and all four patients are alive with no evidence of recurrent disease with 6+ to 54+ months' follow-up. This appears to be the only report of N-myc analysis in this group of children. It would be interesting to analyze more neuroblastomas from patients who present with opsoclonus and myoclonus to determine how many of these patients have single N-myc copy tumors.

Functional properties of proteins coded by three human alpha-interferon genes and a pseudogene.

We have characterized the functional properties of four highly purified recombinant human class I alpha-interferon subtypes whose biological activities have not been described previously. We selected biological and biochemical activities that may discriminate between different functions of these molecules. We found that the alpha subtypes could be discriminated only by antiviral-host range specificity and natural killer cell activation. Differences in their antiproliferative activity were cell line dependent. Competitive binding, antiproliferative activity in agar, enhancement of expression of HLA-ABC, elevation of 2'-5'-oligoadenylate synthetase levels and enhancement of phosphorylation of the Mr 69,000 protein kinase did not allow discrimination among the alpha I subtypes on the tested cell lines.

Human T-cell lymphotropic virus type I-associated adult T-cell leukemia/lymphoma in an atypical host.

Human T-cell lymphotropic virus type I (HTLV-I) is a unique retrovirus associated with specific malignancies of T cells in humans. The virus is endemic to Japan, the Caribbean, Africa, and the southeastern United States. We report here the first case of HTLV-I-associated lymphoma in an atypical host. The incidence of antibodies in this individual with a T-cell malignancy strongly suggests HTLV-I virus as the causative agent. Direct identification of viral DNA using an HTLV-I-specific probe provides definitive evidence of infection.

N-myc amplification in an infant with stage IVS neuroblastoma.

N-myc amplification is most frequently found in neuroblastoma from patients with stage III and IV disease. Recently a significant association between genomic amplification and poor prognosis has been demonstrated. The primary tumors studied from patients with stage IVS disease have reportedly had a single copy of N-myc, and these patients are alive without progressive disease. We report a patient with stage IVS neuroblastoma with N-myc amplification who developed widespread metastasis within 6 months of diagnosis. The same correlation between oncogene copy number and progressive disease that has been seen in those patients with stage II, III, and IV disease was seen in this patient with stage IVS neuroblastoma.

Establishment and characterization of a neuroendocrine skin carcinoma cell line.

A neuroendocrine skin carcinoma cell line MKL-1 has been established from a nodal metastasis in a 26-year-old patient. The line grows as irregularly outlined, loosely packed floating aggregates lacking central necrosis. MKL-1 is hyperdiploid and has a mean doubling time of 120 hours. Xenografts of 2 X 10(7) MKL-1 cells produce tumors in nude mice at 4 to 6 weeks after subcutaneous inoculation. The xenografts were morphologically indistinguishable from the original skin primary and the nodal metastasis. Electron microscopy revealed sparse membrane-bound neurosecretory granules, and conspicuous, paranuclear aggregates of intermediate filaments. Immunohistochemical study showed diffuse and consistent staining with neuron-specific enolase, while bombesin, adrenocorticotrophic hormone, Leu-enkephalin, substance P, and vasoactive intestinal polypeptide displayed heterogeneous and variable expression. Uniform staining of all cells appearing as cytoplasmic fibrils and paranuclear aggregates was noted with antibodies to cytokeratin. Appreciable amounts of cytokeratin polypeptides 8, 18, and 19 and IT protein were seen on two-dimensional gel electrophoresis of cytoskeletal preparations from MKL-1 cells and from tumor-rich frozen sections. Immunostaining also showed coexpression of neurofilaments arranged in paranuclear aggregates; gel electrophoresis and immunoblotting demonstrated the presence in MKL-1 cells of prominent amounts of the small neurofilament polypeptide. Focal expression of desmoplakin was noted in the xenografts. The cells reacted with monoclonal antibodies anti-Leu-7 and anti-Leu-M1 but did not react with antibodies to human lymphocyte antigens (HLA)-A, HLA-B, and HLA-C. Cytogenetic analysis revealed the presence of 3 chromosomally abnormal cell lines with the majority of metaphase cells demonstrating a gain of an isochromosome of the short arm of chromosome 5. Thus, MKL-1 cell line shares several characteristics with small cell neuroendocrine bronchopulmonary carcinoma cell lines but shows distinct cytogenetic abnormalities.