Inhibition of major histocompatibility complex-I antigen presentation by sarbecovirus ORF7a proteins.

Viruses employ a variety of strategies to escape or counteract immune responses, including depletion of cell surface major histocompatibility complex class I (MHC-I), that would ordinarily present viral peptides to CD8+ cytotoxic T cells. As part of a screen to elucidate biological activities associated with individual severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) viral proteins, we found that ORF7a reduced cell surface MHC-I levels by approximately fivefold. Nevertheless, in cells infected with SARS-CoV-2, surface MHC-I levels were reduced even in the absence of ORF7a, suggesting additional mechanisms of MHC-I down-regulation. ORF7a proteins from a sample of sarbecoviruses varied in their ability to induce MHC-I down-regulation and, unlike SARS-CoV-2, the ORF7a protein from SARS-CoV lacked MHC-I downregulating activity. A single amino acid at position 59 (T/F) that is variable among sarbecovirus ORF7a proteins governed the difference in MHC-I downregulating activity. SARS-CoV-2 ORF7a physically associated with the MHC-I heavy chain and inhibited the presentation of expressed antigen to CD8+ T cells. Specifically, ORF7a prevented the assembly of the MHC-I peptide loading complex and caused retention of MHC-I in the endoplasmic reticulum. The differential ability of ORF7a proteins to function in this way might affect sarbecovirus dissemination and persistence in human populations, particularly those with infection- or vaccine-elicited immunity.

A viral arsenal.

SARS-CoV-2 wields versatile proteins to foil our immune system's counterattack.

SARS-CoV-2 prefusion spike protein stabilized by six rather than two prolines is more potent for inducing antibodies that neutralize viral variants of concern.

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the main target for neutralizing antibodies (NAbs). The S protein trimer is anchored in the virion membrane in its prefusion (preS) but metastable form. The preS protein has been stabilized by introducing two or six proline substitutions, to generate stabilized, soluble 2P or HexaPro (6P) preS proteins. Currently, it is not known which form is the most immunogenic. Here, we generated recombinant vesicular stomatitis virus (rVSV) expressing preS-2P, preS-HexaPro, and native full-length S, and compared their immunogenicity in mice and hamsters. The rVSV-preS-HexaPro produced and secreted significantly more preS protein compared to rVSV-preS-2P. Importantly, rVSV-preS-HexaPro triggered significantly more preS-specific serum IgG antibody than rVSV-preS-2P in both mice and hamsters. Antibodies induced by preS-HexaPro neutralized the B.1.1.7, B.1.351, P.1, B.1.427, and B.1.617.2 variants approximately two to four times better than those induced by preS-2P. Furthermore, preS-HexaPro induced a more robust Th1-biased cellular immune response than preS-2P. A single dose (104 pfu) immunization with rVSV-preS-HexaPro and rVSV-preS-2P provided complete protection against challenge with mouse-adapted SARS-CoV-2 and B.1.617.2 variant, whereas rVSV-S only conferred partial protection. When the immunization dose was lowered to 103 pfu, rVSV-preS-HexaPro induced two- to sixfold higher antibody responses than rVSV-preS-2P in hamsters. In addition, rVSV-preS-HexaPro conferred 70% protection against lung infection whereas only 30% protection was observed in the rVSV-preS-2P. Collectively, our data demonstrate that both preS-2P and preS-HexaPro are highly efficacious but preS-HexaPro is more immunogenic and protective, highlighting the advantages of using preS-HexaPro in the next generation of SARS-CoV-2 vaccines.

Principles and practical applications of structure-based vaccine design.

Viral proteins fold into a variety of structures as they perform their functions. Structure-based vaccine design aims to exploit knowledge of an antigen's architecture to stabilize it in a vulnerable conformation. We summarize the general principles of structure-based vaccine design, with a focus on the major types of sequence modifications: proline, disulfide, cavity-filling, electrostatic and hydrogen-bond substitution, as well as domain deletion. We then review recent applications of these principles to vaccine-design efforts across five viral families: Coronaviridae, Orthomyxoviridae, Paramyxoviridae, Pneumoviridae, and Filoviridae. Outstanding challenges include continued application of proven design principles to pathogens of interest, as well as development of new strategies for those pathogens that resist traditional techniques.

Predicted impact of the viral mutational landscape on the cytotoxic response against SARS-CoV-2.

The massive assessment of immune evasion due to viral mutations that increase COVID-19 susceptibility can be computationally facilitated. The adaptive cytotoxic T response is critical during primary infection and the generation of long-term protection. Here, potential HLA class I epitopes in the SARS-CoV-2 proteome were predicted for 2,915 human alleles of 71 families using the netMHCIpan EL algorithm. Allele families showed extreme epitopic differences, underscoring genetic variability of protective capacity between humans. Up to 1,222 epitopes were associated with any of the twelve supertypes, that is, allele clusters covering 90% population. Next, from all mutations identified in ~118,000 viral NCBI isolates, those causing significant epitope score reduction were considered epitope escape mutations. These mutations mainly involved non-conservative substitutions at the second and C-terminal position of the ligand core, or total ligand removal by large recurrent deletions. Escape mutations affected 47% of supertype epitopes, which in 21% of cases concerned isolates from two or more sub-continental areas. Some of these changes were coupled, but never surpassed 15% of evaded epitopes for the same supertype in the same isolate, except for B27. In contrast to most supertypes, eight allele families mostly contained alleles with few SARS-CoV-2 ligands. Isolates harboring cytotoxic escape mutations for these families co-existed geographically within sub-Saharan and Asian populations enriched in these alleles according to the Allele Frequency Net Database. Collectively, our findings indicate that escape mutation events have already occurred for half of HLA class I supertype epitopes. However, it is presently unlikely that, overall, it poses a threat to the global population. In contrast, single and double mutations for susceptible alleles may be associated with viral selective pressure and alarming local outbreaks. The integration of genomic, geographical and immunoinformatic information eases the surveillance of variants potentially affecting the global population, as well as minority subpopulations.

CRESSP: a comprehensive pipeline for prediction of immunopathogenic SARS-CoV-2 epitopes using structural properties of proteins.

The development of autoimmune diseases following SARS-CoV-2 infection, including multisystem inflammatory syndrome, has been reported, and several mechanisms have been suggested, including molecular mimicry. We developed a scalable, comparative immunoinformatics pipeline called cross-reactive-epitope-search-using-structural-properties-of-proteins (CRESSP) to identify cross-reactive epitopes between a collection of SARS-CoV-2 proteomes and the human proteome using the structural properties of the proteins. Overall, by searching 4 911 245 proteins from 196 352 SARS-CoV-2 genomes, we identified 133 and 648 human proteins harboring potential cross-reactive B-cell and CD8+ T-cell epitopes, respectively. To demonstrate the robustness of our pipeline, we predicted the cross-reactive epitopes of coronavirus spike proteins, which were recognized by known cross-neutralizing antibodies. Using single-cell expression data, we identified PARP14 as a potential target of intermolecular epitope spreading between the virus and human proteins. Finally, we developed a web application (https://ahs2202.github.io/3M/) to interactively visualize our results. We also made our pipeline available as an open-source CRESSP package (https://pypi.org/project/cressp/), which can analyze any two proteomes of interest to identify potentially cross-reactive epitopes between the proteomes. Overall, our immunoinformatic resources provide a foundation for the investigation of molecular mimicry in the pathogenesis of autoimmune and chronic inflammatory diseases following COVID-19.

HLA-C*04:01 Affects HLA Class I Heterozygosity and Predicted Affinity to SARS-CoV-2 Peptides, and in Combination With Age and Sex of Armenian Patients Contributes to COVID-19 Severity.

The novel SARS-CoV-2 coronavirus infection has become a global health concern, causing the COVID-19 pandemic. The disease symptoms and outcomes depend on the host immunity, in which the human leukocyte antigen (HLA) molecules play a distinct role. The HLA alleles have an inter-population variability, and understanding their link to the COVID-19 in an ethnically distinct population may contribute to personalized medicine. The present study aimed at detecting associations between common HLA alleles and COVID-19 susceptibility and severity in Armenians. In 299 COVID-19 patients (75 asymptomatic, 102 mild/moderate, 122 severe), the association between disease severity and classic HLA-I and II loci was examined. We found that the advanced age, male sex of patients, and sex and age interaction significantly contributed to the severity of the disease. We observed that an age-dependent effect of HLA-B*51:01 carriage [odds ratio (OR)=0.48 (0.28-0.80), Pbonf <0.036] is protective against severe COVID-19. Contrary, the HLA-C*04:01 allele, in a dose-dependent manner, was associated with a significant increase in the disease severity [OR (95% CI) =1.73 (1.20-2.49), Pbonf <0.021] and an advancing age (P<0.013). The link between HLA-C*04:01 and age was secondary to a stronger association between HLA-C*04:01 and disease severity. However, HLA-C*04:01 exerted a sex-dependent differential distribution between clinical subgroups [females: P<0.0012; males: P=0.48]. The comparison of HLA-C*04:01 frequency between subgroups and 2,781 Armenian controls revealed a significant incidence of HLA-C*04:01 deficiency in asymptomatic COVID-19. HLA-C*04:01 homozygous genotype in patients blueprinted a decrease in heterozygosity of HLA-B and HLA class-I loci. In HLA-C*04:01 carriers, these changes translated to the SARS-CoV-2 peptide presentation predicted inefficacy by HLA-C and HLA class-I molecules, simultaneously enhancing the appropriate HLA-B potency. In patients with clinical manifestation, due to the high prevalence of HLA-C*04:01, these effects provided a decrease of the HLA class-I heterozygosity and an ability to recognize SARS-CoV-2 peptides. Based on our observations, we developed a prediction model involving demographic variables and HLA-C*04:01 allele for the identification of potential cases with the risk of hospitalization (the area under the curve (AUC) = 86.2%) or severe COVID-19 (AUC =71%).

Induction of Hepatitis E Virus Anti-ORF3 Antibodies from Systemic Administration of a Muscle-Specific Adeno-Associated Virus (AAV) Vector.

The hepatitis E virus (HEV) is a major global health problem, leading to large outbreaks in the developing world and chronic infections in the developed world. HEV is a non-enveloped virus, which circulates in the blood in a quasi-enveloped form. The quasi-envelope protects HEV particles from neutralising anti-capsid antibodies in the serum; however, most vaccine approaches are designed to induce an immune response against the HEV capsid. In this study, we explored systemic in vivo administration of a novel synthetic and myotropic Adeno-associated virus vector (AAVMYO3) to express the small HEV phosphoprotein ORF3 (found on quasi-enveloped HEV) in the musculature of mice, resulting in the robust and dose-dependent formation of anti-ORF3 antibodies. Neutralisation assays using the serum of ORF3 AAV-transduced mice showed a modest inhibitory effect on the infection of quasi-enveloped HEV in vivo, comparable to previously characterised anti-ORF3 antibodies used as a control. The novel AAVMYO3 capsid used in this study can serve as a versatile platform for the continued development of vector-based vaccines against HEV and other infectious agents, which could complement traditional vaccines akin to the current positive experience with SARS-CoV-2.

Rapid Biosensor of SARS-CoV-2 Using Specific Monoclonal Antibodies Recognizing Conserved Nucleocapsid Protein Epitopes.

Coronavirus disease 2019 (COVID-19), the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is characterized by symptoms such as fever, fatigue, a sore throat, diarrhea, and coughing. Although various new vaccines against COVID-19 have been developed, early diagnostics, isolation, and prevention remain important due to virus mutations resulting in rapid and high disease transmission. Amino acid substitutions in the major diagnostic target antigens of SARS-CoV-2 may lower the sensitivity for the detection of SARS-CoV-2. For this reason, we developed specific monoclonal antibodies that bind to epitope peptides as antigens for the rapid detection of SARS-CoV-2 NP. The binding affinity between antigenic peptides and monoclonal antibodies was investigated, and a sandwich pair for capture and detection was employed to develop a rapid biosensor for SARS-CoV-2 NP. The rapid biosensor, based on a monoclonal antibody pair binding to conserved epitopes of SARS-CoV-2 NP, detected cultured virus samples of SARS-CoV-2 (1.4 × 103 TCID50/reaction) and recombinant NP (1 ng/mL). Laboratory confirmation of the rapid biosensor was performed with clinical specimens (n = 16) from COVID-19 patients and other pathogens. The rapid biosensor consisting of a monoclonal antibody pair (75E12 for capture and the 54G6/54G10 combination for detection) binding to conserved epitopes of SARS-CoV-2 NP could assist in the detection of SARS-CoV-2 NP under the circumstance of spreading SARS-CoV-2 variants.

Cross-reactive memory T cells associate with protection against SARS-CoV-2 infection in COVID-19 contacts.

Cross-reactive immune responses to SARS-CoV-2 have been observed in pre-pandemic cohorts and proposed to contribute to host protection. Here we assess 52 COVID-19 household contacts to capture immune responses at the earliest timepoints after SARS-CoV-2 exposure. Using a dual cytokine FLISpot assay on peripheral blood mononuclear cells, we enumerate the frequency of T cells specific for spike, nucleocapsid, membrane, envelope and ORF1 SARS-CoV-2 epitopes that cross-react with human endemic coronaviruses. We observe higher frequencies of cross-reactive (p = 0.0139), and nucleocapsid-specific (p = 0.0355) IL-2-secreting memory T cells in contacts who remained PCR-negative despite exposure (n = 26), when compared with those who convert to PCR-positive (n = 26); no significant difference in the frequency of responses to spike is observed, hinting at a limited protective function of spike-cross-reactive T cells. Our results are thus consistent with pre-existing non-spike cross-reactive memory T cells protecting SARS-CoV-2-naïve contacts from infection, thereby supporting the inclusion of non-spike antigens in second-generation vaccines.

Identification of immunodominant epitopes on nucleocapsid and spike proteins of the SARS-CoV-2 in Iranian COVID-19 patients.

Given the emergence of SARS-CoV-2 virus as a life-threatening pandemic, identification of immunodominant epitopes of the viral structural proteins, particularly the nucleocapsid (NP) protein and receptor-binding domain (RBD) of spike protein, is important to determine targets for immunotherapy and diagnosis. In this study, epitope screening was performed using a panel of overlapping peptides spanning the entire sequences of the RBD and NP proteins of SARS-CoV-2 in the sera from 66 COVID-19 patients and 23 healthy subjects by enzyme-linked immunosorbent assay (ELISA). Our results showed that while reactivity of patients' sera with reduced recombinant RBD protein was significantly lower than the native form of RBD (P < 0.001), no significant differences were observed for reactivity of patients' sera with reduced and non-reduced NP protein. Pepscan analysis revealed weak to moderate reactivity towards different RBD peptide pools, which was more focused on peptides encompassing amino acids (aa) 181-223 of RBD. NP peptides, however, displayed strong reactivity with a single peptide covering aa 151-170. These findings were confirmed by peptide depletion experiments using both ELISA and western blotting. Altogether, our data suggest involvement of mostly conformational disulfide bond-dependent immunodominant epitopes in RBD-specific antibody response, while the IgG response to NP is dominated by linear epitopes. Identification of dominant immunogenic epitopes in NP and RBD of SARS-CoV-2 could provide important information for the development of passive and active immunotherapy as well as diagnostic tools for the control of COVID-19 infection.

Crystal Structures of Bat and Human Coronavirus ORF8 Protein Ig-Like Domain Provide Insights Into the Diversity of Immune Responses.

ORF8 is a viral immunoglobulin-like (Ig-like) domain protein encoded by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA genome. It tends to evolve rapidly and interfere with immune responses. However, the structural characteristics of various coronavirus ORF8 proteins and their subsequent effects on biological functions remain unclear. Herein, we determined the crystal structures of SARS-CoV-2 ORF8 (S84) (one of the epidemic isoforms) and the bat coronavirus RaTG13 ORF8 variant at 1.62 Å and 1.76 Å resolution, respectively. Comparison of these ORF8 proteins demonstrates that the 62-77 residues in Ig-like domain of coronavirus ORF8 adopt different conformations. Combined with mutagenesis assays, the residue Cys20 of ORF8 is responsible for forming the covalent disulfide-linked dimer in crystal packing and in vitro biochemical conditions. Furthermore, immune cell-binding assays indicate that various ORF8 (SARS-CoV-2 ORF8 (L84), ORF8 (S84), and RaTG13 ORF8) proteins have different interaction capabilities with human CD14+ monocytes in human peripheral blood. These results provide new insights into the specific characteristics of various coronavirus ORF8 and suggest that ORF8 variants may influence disease-related immune responses.

Super.Complex: A supervised machine learning pipeline for molecular complex detection in protein-interaction networks.

Characterization of protein complexes, i.e. sets of proteins assembling into a single larger physical entity, is important, as such assemblies play many essential roles in cells such as gene regulation. From networks of protein-protein interactions, potential protein complexes can be identified computationally through the application of community detection methods, which flag groups of entities interacting with each other in certain patterns. Most community detection algorithms tend to be unsupervised and assume that communities are dense network subgraphs, which is not always true, as protein complexes can exhibit diverse network topologies. The few existing supervised machine learning methods are serial and can potentially be improved in terms of accuracy and scalability by using better-suited machine learning models and parallel algorithms. Here, we present Super.Complex, a distributed, supervised AutoML-based pipeline for overlapping community detection in weighted networks. We also propose three new evaluation measures for the outstanding issue of comparing sets of learned and known communities satisfactorily. Super.Complex learns a community fitness function from known communities using an AutoML method and applies this fitness function to detect new communities. A heuristic local search algorithm finds maximally scoring communities, and a parallel implementation can be run on a computer cluster for scaling to large networks. On a yeast protein-interaction network, Super.Complex outperforms 6 other supervised and 4 unsupervised methods. Application of Super.Complex to a human protein-interaction network with ~8k nodes and ~60k edges yields 1,028 protein complexes, with 234 complexes linked to SARS-CoV-2, the COVID-19 virus, with 111 uncharacterized proteins present in 103 learned complexes. Super.Complex is generalizable with the ability to improve results by incorporating domain-specific features. Learned community characteristics can also be transferred from existing applications to detect communities in a new application with no known communities. Code and interactive visualizations of learned human protein complexes are freely available at: https://sites.google.com/view/supercomplex/super-complex-v3-0.

A virus-specific monocyte inflammatory phenotype is induced by SARS-CoV-2 at the immune-epithelial interface.

Infection by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) provokes a potentially fatal pneumonia with multiorgan failure, and high systemic inflammation. To gain mechanistic insight and ferret out the root of this immune dysregulation, we modeled, by in vitro coculture, the interactions between infected epithelial cells and immunocytes. A strong response was induced in monocytes and B cells, with a SARS-CoV-2-specific inflammatory gene cluster distinct from that seen in influenza A or Ebola virus-infected cocultures, and which reproduced deviations reported in blood or lung myeloid cells from COVID-19 patients. A substantial fraction of the effect could be reproduced after individual transfection of several SARS-CoV-2 proteins (Spike and some nonstructural proteins), mediated by soluble factors, but not via transcriptional induction. This response was greatly muted in monocytes from healthy children, perhaps a clue to the age dependency of COVID-19. These results suggest that the inflammatory malfunction in COVID-19 is rooted in the earliest perturbations that SARS-CoV-2 induces in epithelia.

A vesicular stomatitis virus-based prime-boost vaccination strategy induces potent and protective neutralizing antibodies against SARS-CoV-2.

The development of safe and effective vaccines to prevent SARS-CoV-2 infections remains an urgent priority worldwide. We have used a recombinant vesicular stomatitis virus (rVSV)-based prime-boost immunization strategy to develop an effective COVID-19 vaccine candidate. We have constructed VSV genomes carrying exogenous genes resulting in the production of avirulent rVSV carrying the full-length spike protein (SF), the S1 subunit, or the receptor-binding domain (RBD) plus envelope (E) protein of SARS-CoV-2. Adding the honeybee melittin signal peptide (msp) to the N-terminus enhanced the protein expression, and adding the VSV G protein transmembrane domain and the cytoplasmic tail (Gtc) enhanced protein incorporation into pseudotype VSV. All rVSVs expressed three different forms of SARS-CoV-2 spike proteins, but chimeras with VSV-Gtc demonstrated the highest rVSV-associated expression. In immunized mice, rVSV with chimeric S protein-Gtc derivatives induced the highest level of potent neutralizing antibodies and T cell responses, and rVSV harboring the full-length msp-SF-Gtc proved to be the superior immunogen. More importantly, rVSV-msp-SF-Gtc vaccinated animals were completely protected from a subsequent SARS-CoV-2 challenge. Overall, we have developed an efficient strategy to induce a protective response in SARS-CoV-2 challenged immunized mice. Vaccination with our rVSV-based vector may be an effective solution in the global fight against COVID-19.

Deep dissection of the antiviral immune profile of patients with COVID-19.

In light of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants potentially undermining humoral immunity, it is important to understand the fine specificity of the antiviral antibodies. We screened 20 COVID-19 patients for antibodies against 9 different SARS-CoV-2 proteins observing responses against the spike (S) proteins, the receptor-binding domain (RBD), and the nucleocapsid (N) protein which were of the IgG1 and IgG3 subtypes. Importantly, mutations which typically occur in the B.1.351 "South African" variant, significantly reduced the binding of anti-RBD antibodies. Nine of 20 patients were critically ill and were considered high-risk (HR). These patients showed significantly higher levels of transforming growth factor beta (TGF-ß) and myeloid-derived suppressor cells (MDSC), and lower levels of CD4+ T cells expressing LAG-3 compared to standard-risk (SR) patients. HR patients evidenced significantly higher anti-S1/RBD IgG antibody levels and an increased neutralizing activity. Importantly, a large proportion of S protein-specific antibodies were glycosylation-dependent and we identified a number of immunodominant linear epitopes within the S1 and N proteins. Findings derived from this study will not only help us to identify the most relevant component of the anti-SARS-CoV-2 humoral immune response but will also enable us to design more meaningful immunomonitoring methods for anti-COVID-19 vaccines.

Response kinetics of different classes of antibodies to SARS-CoV2 infection in the Japanese population: The IgA and IgG titers increased earlier than the IgM titers.

To better understand the immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in individuals with COVID-19, it is important to investigate the kinetics of the antibody responses and their associations with the clinical course in different populations, since there seem to be considerable differences between Western and Asian populations in the clinical features and spread of COVID-19. In this study, we serially measured the serum titers of IgM, IgG and IgA antibodies generated against the nucleocapsid protein (NCP), S1 subunit of the spike protein (S1), and receptor-binding domain in the S1 subunit (RBD) of SARS-CoV-2 in Japanese individuals with COVID-19. Among the IgM, IgG, and IgA antibodies, IgA antibodies against all of the aforementioned viral proteins were the first to appear after the infection, and IgG and/or IgA seroconversion often preceded IgM seroconversion. In regard to the timeline of the antibody responses to the different viral proteins (NCP, S1 and RBD), IgA against NCP appeared than IgA against S1 or RBD, while IgM and IgG against S1 appeared earlier than IgM/IgG against NCP or RBD. The IgG responses to all three viral proteins and responses of all three antibody classes to S1 and RBD were sustained for longer durations than the IgA/IgM responses to all three viral proteins and responses of all three antibody classes to NCP, respectively. The seroconversion of IgA against NCP occurred later and less frequently in patients with mild COVID-19. These results suggest possible differences in the antibody responses to SARS-CoV-2 antigens between the Japanese and Western populations.

Enhanced High Mutation Rate and Natural Selection to Produce Attenuated Viral Vaccine with CRISPR Toolkit in RNA Viruses especially SARS-CoV-2.

The best and most effective way to combat pandemics is to use effective vaccines and live attenuated vaccines are among the most effective vaccines. However, one of the major problems is the length of time it takes to get the attenuated vaccines. Today, the CRISPR toolkit (Clustered Regularly Inerspaced Short Palindromic Repeats) has made it possible to make changes with high efficiency and speed. Using this toolkit to make point mutations on the RNA virus's genome in a coculture of permissive and nonpermissive cells and under controlled conditions can accelerate changes in the genome and accelerate natural selection to obtain live attenuated vaccines.

Advances and insights in the diagnosis of viral infections.

Viral infections are the most common among diseases that globally require around 60 percent of medical care. However, in the heat of the pandemic, there was a lack of medical equipment and inpatient facilities to provide all patients with viral infections. The detection of viral infections is possible in three general ways such as (i) direct virus detection, which is performed immediately 1-3 days after the infection, (ii) determination of antibodies against some virus proteins mainly observed during/after virus incubation period, (iii) detection of virus-induced disease when specific tissue changes in the organism. This review surveys some global pandemics from 1889 to 2020, virus types, which induced these pandemics, and symptoms of some viral diseases. Non-analytical methods such as radiology and microscopy also are overviewed. This review overlooks molecular analysis methods such as nucleic acid amplification, antibody-antigen complex determination, CRISPR-Cas system-based viral genome determination methods. Methods widely used in the certificated diagnostic laboratory for SARS-CoV-2, Influenza A, B, C, HIV, and other viruses during a viral pandemic are outlined. A comprehensive overview of molecular analytical methods has shown that the assay's sensitivity, accuracy, and suitability for virus detection depends on the choice of the number of regions in the viral open reading frame (ORF) genome sequence and the validity of the selected analytical method.

Influenza vaccination in the elderly boosts antibodies against conserved viral proteins and egg-produced glycans.

Seasonal influenza vaccination elicits a diminished adaptive immune response in the elderly, and the mechanisms of immunosenescence are not fully understood. Using Ig-Seq, we found a marked increase with age in the prevalence of cross-reactive (CR) serum antibodies that recognize both the H1N1 (vaccine-H1) and H3N2 (vaccine-H3) components of an egg-produced split influenza vaccine. CR antibodies accounted for 73% ± 18% of the serum vaccine responses in a cohort of elderly donors, 65% ± 15% in late middle-aged donors, and only 13% ± 5% in persons under 35 years of age. The antibody response to non-HA antigens was boosted by vaccination. Recombinant expression of 19 vaccine-H1+H3 CR serum monoclonal antibodies (s-mAbs) revealed that they predominantly bound to non-HA influenza proteins. A sizable fraction of vaccine-H1+H3 CR s-mAbs recognized with high affinity the sulfated glycans, in particular sulfated type 2 N-acetyllactosamine (Galß1-4GalNAcß), which is found on egg-produced proteins and thus unlikely to contribute to protection against influenza infection in humans. Antibodies against sulfated glycans in egg-produced vaccine had been identified in animals but were not previously characterized in humans. Collectively, our results provide a quantitative basis for how repeated exposure to split influenza vaccine correlates with unintended focusing of serum antibody responses to non-HA antigens that may result in suboptimal immunity against influenza.