Durability of SARS-CoV-2 Specific IgG Antibody Responses Following Two Doses of Match and Mixed COVID-19 Vaccines Regimens in Saudi Population.

Background: SARS-CoV-2 pandemic continues to threaten the human population with millions of infections and deaths worldwide. Vaccination campaigns undertaken by several countries have resulted in a notable decrease in hospitalization and deaths. However, with the emergence of new virus variants, it is critical to determine the longevity and the protection efficiency provided by the current authorized vaccines. Aim: The aims of this study are to provide data about the magnitude of immune responses in individuals fully vaccinated against COVID-19 in Riyadh province of Saudi Arabia. Also, to evaluate the continuity of specific IgG levels and compare the titers in individuals who have been received two doses of the matched and mixed vaccines, including Pfizer and AstraZeneca against SARS-CoV-2 during the period of three to six months. Moreover, we analyze the current state of immune response in terms of antibody responses in thepopulation postvaccination using homogenous or hetrogenous vaccine regimen. Methods: A total of 141 healthy volunteers were recruited to our study; blood (n=63) and the saliva samples (n=78) and were collected from fully vaccinated individuals in Riyadh city. We employed a specific ELISA assay in plasma and saliva of fully vaccinated individuals. Results: IgG levels varied with age groups with the highest concentration in the age group 19-29 years, but the age group (≥50) had the lowest IgG concentration. The IgG levels in both serum and saliva were higher after three months and start to wane after six months. Individuals who received mixed types of vaccines had significantly better response than Pfizer vaccine alone. Conclusion: The current study investigates the status of humoral responses in different age groups, in terms of antibody measurements. These data will help to evaluate the need for further COVID-19 vaccine doses and to what extent a two-dose regimen will protect vaccinated individuals.

Development and Validation of Rapid In-House Diagnostic ELISA Kits for Detection of Human Orthopneumovirus in Clinical Samples.

Currently, the standard assay employed to diagnose human orthopneumovirus infection is real-time reverse transcriptase PCR assay (rRT-PCR), a costly and time-consuming procedure that requires the manipulation of infectious viruses. In addition to RT-PCR, serological tests can complement the molecular diagnostic methods and have proven to be important tools in sero-surveillance. In this study, we report the development, optimization, and validation of a novel and rapid in-house diagnostic ELISA kit to detect human orthopneumovirus in clinical samples. We developed three sensitive ELISA formats through the immunization of rats with novel recombinant pPOE-F or pPOE-TF vectors. The two vectors expressed either the full-length (pPOE-F) or the truncated form (pPOE-TF) of the fusion (F) protein. The developed ELISA kits were optimized for coating buffer, capture antibody, blocking buffer, sample antigen, detection antibodies, and peroxidase-conjugated antibody, and validated using 75 rRT-PCR-confirmed nasopharyngeal aspirate (NPA) human orthopneumovirus samples and 25 negative samples collected from hospitalized children during different epidemic seasons between 2014 and 2017. Our results indicate that rats immunized with pPOE-F or pPOE-TF showed significant induction of high levels of MPAs. Validation of the ELISA method was compared to the rRT-PCR and the sensitivity hierarchy of these developed ELISA assays was considered from highest to lowest: indirect competitive inhibition ELISA (93.3%) > indirect antigen-capture ELISA (90.6%) > direct antigen-capture ELISA (86.6%). The development of the rapid in-house diagnostic ELISA kits described in this study demonstrates that a specific, rapid and sensitive test for human orthopneumovirus antigens could be successfully applied to samples collected from hospitalized children during different epidemics and can help in the efficient diagnosis of respiratory syncytial viral infections.

SARS-CoV-2: An Overview of Virus Genetics, Transmission, and Immunopathogenesis.

The human population is currently facing the third and possibly the worst pandemic caused by human coronaviruses (CoVs). The virus was first reported in Wuhan, China, on 31 December 2019 and spread within a short time to almost all countries of the world. Genome analysis of the early virus isolates has revealed high similarity with SARS-CoV and hence the new virus was officially named SARS-CoV-2. Since CoVs have the largest genome among all RNA viruses, they can adapt to many point mutation and recombination events; particularly in the spike gene, which enable these viruses to rapidly change and evolve in nature. CoVs are known to cross the species boundaries by using different cellular receptors. Both animal reservoir and intermediate host for SARS-CoV-2 are still unresolved and necessitate further investigation. In the current review, different aspects of SARS-CoV-2 biology and pathogenicity are discussed, including virus genetics and evolution, spike protein and its role in evolution and adaptation to novel hosts, and virus transmission and persistence in nature. In addition, the immune response developed during SARS-CoV-2 infection is demonstrated with special reference to the interplay between immune cells and their role in disease progression. We believe that the SARS-CoV-2 outbreak will not be the last and spillover of CoVs from bats will continue. Therefore, establishing intervention approaches to reduce the likelihood of future CoVs spillover from natural reservoirs is a priority.

Sequence and phylogentic analysis of MERS-CoV in Saudi Arabia, 2012-2019.

BACKGROUND: The Middle East Respiratory Syndrome-related Coronavirus (MERS-CoV) continues to exist in the Middle East sporadically. Thorough investigations of the evolution of human coronaviruses (HCoVs) are urgently required. In the current study, we studied amplified fragments of ORF1a/b, Spike (S) gene, ORF3/4a, and ORF4b of four human MERS-CoV strains for tracking the evolution of MERS-CoV over time. METHODS: RNA isolated from nasopharyngeal aspirate, sputum, and tracheal swabs/aspirates from hospitalized patients with suspected MERS-CoV infection were analyzed for amplification of nine variable genomic fragments. Sequence comparisons were done using different bioinformatics tools available. RESULTS: Several mutations were identified in ORF1a/b, ORF3/4a and ORF4b, with the highest mutation rates in the S gene. Five codons; 4 in ORF1a and 1 in the S gene, were found to be under selective pressure. Characteristic amino acid changes, potentially hosted and year specific were defined across the S protein and in the receptor-binding domain Phylogenetic analysis using S gene sequence revealed clustering of MERS-CoV strains into three main clades, A, B and C with subdivision of with clade B into B1 to B4. CONCLUSIONS: In conclusion, MERS-CoV appears to continuously evolve. It is recommended that the molecular and pathobiological characteristics of future MERS-CoV strains should be analyzed on regular basis to prevent potential future outbreaks at early phases.

Induction of Immune Responses and Immune Evasion by Human Bocavirus.

Respiratory tract infections are the primary cause of morbidity and mortality globally. Human bocavirus 1 (HBoV1), a member of the Parvoviridae family causes a wide spectrum of respiratory diseases in children, and gastroenteritis in adults. The mechanisms of latency, persistence, and reinfection of Bocavirus are poorly understood at present due to the lack of permissive cell lines and efficient animal models. Moreover, the dual infections of HBoV and other respiratory viruses further complicate the study of the pathogenicity of Bocaviruses. The data on immunological consequences of Bocavirus infection are sparse. However, the existing data have highlighted the role of CD4 T cells in Bocavirus infection. High titres of HBoV-specific antibodies have been detected in different populations suggesting its ubiquitous prevalence. Interestingly, the mechanism employed by Bocavirus to evade the immune system mostly targets type I IFN pathways and cause pyroptotic cell death of host cells. This review summarizes the immune responses evoked in response to Bocavirus infection, escape mechanism employed by the virus, and the vaccination strategies, including antisense technology to combat Bocavirus infections.

Double-edged role of natural killer cells during RSV infection.

Natural killer cells play a vital role in the rejection of tumors and pathogen-infected cells. NK cells are indispensable in the early immune response against viral infections by directly targeting infected cells. Furthermore, NK cells influence adaptive immunity by driving virus-specific T-cell responses. Respiratory syncytial virus, a highly contagious virus that causes bronchiolitis, is the main reason for mortality in infants and elderly patients. RSV infection triggers both innate and adaptive immune responses. However, immunity against RSV is ephemeral due to the impaired development of immunological memory. The role of NK cells during RSV infection remains ambiguous. NK cells play a dual role in RSV infection; initially, their role is a protective one as they utilize their intrinsic cytotoxicity, followed by a detrimental one that induces lung injury due to the inhibition of antibody responses and the secretion of pro-inflammatory factors. Noteworthy, IFN-γ released from NK cells play a critical role in promoting a shift to adaptive responses and inhibiting antibody responses in neonates. Indeed, NK cells have a pro-inflammatory and inhibitory role rather than a cytotoxic one that contributes to the severity of the disease. Therapeutic options, including DNA-protein-based vaccines, synthetic peptides, and attenuated strains, are presently under tests. However, there is a need for effective strategies to augment NK cell activity and circumvent the pro-inflammatory activity to benefit the host. In this review, we focused on the role played by NK cells in the immune response and its outcome on the immunopathogenesis of RSV disease.

Oncolytic H-1 Parvovirus Shows Safety and Signs of Immunogenic Activity in a First Phase I/IIa Glioblastoma Trial.

Oncolytic virotherapy may be a means of improving the dismal prognosis of malignant brain tumors. The rat H-1 parvovirus (H-1PV) suppresses tumors in preclinical glioma models, through both direct oncolysis and stimulation of anticancer immune responses. This was the basis of ParvOryx01, the first phase I/IIa clinical trial of an oncolytic parvovirus in recurrent glioblastoma patients. H-1PV (escalating dose) was administered via intratumoral or intravenous injection. Tumors were resected 9 days after treatment, and virus was re-administered around the resection cavity. Primary endpoints were safety and tolerability, virus distribution, and maximum tolerated dose (MTD). Progression-free and overall survival and levels of viral and immunological markers in the tumor and peripheral blood were also investigated. H-1PV treatment was safe and well tolerated, and no MTD was reached. The virus could cross the blood-brain/tumor barrier and spread widely through the tumor. It showed favorable pharmacokinetics, induced antibody formation in a dose-dependent manner, and triggered specific T cell responses. Markers of virus replication, microglia/macrophage activation, and cytotoxic T cell infiltration were detected in infected tumors, suggesting that H-1PV may trigger an immunogenic stimulus. Median survival was extended in comparison with recent meta-analyses. Altogether, ParvOryx01 results provide an impetus for further H-1PV clinical development.

Co-Activation of Cultured Human Natural Killer Cells: Enhanced Function and Decreased Inhibition.

Natural killer (NK) cells are important immune effector cells that protect the organism against viral infections and cancer. The cytotoxic activity of NK cells is induced by the engagement of a number of different activating surface receptors and controlled by inhibitory receptors to ensure self-tolerance. Resting NK cells need to be co-activated by involvement of at least two distinct activating receptors in order to induce their functional activity. However, in cultured NK cells, which have been expanded in cytokines such as interleukin (IL)-2, the engagement of a single activating receptor may be sufficient to induce their function. Data demonstrated that also cultured NK cells may be co-activated by involvement of certain combinations of activating receptors. This co-activation results in enhanced activation of Vav-1 and ERK signaling pathways and produces greater degranulation. In addition to enhanced functionality, co-activation makes NK cells more resistant to the effect of inhibitory receptors, thereby inducing more potent and efficient NK cell responses.

Antigen-armed antibodies targeting B lymphoma cells effectively activate antigen-specific CD4+ T cells.

The treatment of non-Hodgkin lymphomas has benefited enormously from the introduction of monoclonal antibody-based therapies. However, the efficacy of these treatments varies with lymphoma subtypes and typically decreases with subsequent relapses. Here, we report on antigen-armed antibodies (AgAbs) as a potential treatment of B-cell lymphoma. AgAbs include antigens from ubiquitous pathogens, such as Epstein-Barr virus (EBV), that persist in their host and elicit strong lifelong T-cell responses. They act as vectors by introducing antigen directly into tumor cells to induce an antigen-specific CD4(+) T-cell response against these cells. We have fused antibodies targeting human B-cell surface receptors (CD19-22) to immunodominant T-cell antigens from EBV proteins, including EBNA1, EBNA3B, and EBNA3C. Exposure of EBV-transformed B cells and of Burkitt lymphoma cells to AgAbs led to antigen presentation, T-cell recognition, and target cell killing. The efficiency of AgAb action paralleled the abundance of the targeted molecules on lymphoma cells as well as their HLA class II expression levels. AgAbs can also induce activation and proliferation of EBV-specific memory CD4(+) T cells ex vivo. These studies show the potential of AgAbs as an effective therapeutic strategy against B-cell lymphomas.

Emerging role of Natural killer cells in oncolytic virotherapy.

Natural killer (NK) cells constitute a subtype of lymphocytes that initiate innate immune responses against tumors and virus-infected cells. The ability of NK cells to kill target cells or to produce cytokines depends on the balance between signals from activating and inhibitory cell-surface receptors. Therapies with NK cells involve activation of endogenous NK cells and/or exogenous transfer by hematopoietic stem cell transplantation/adoptive cell therapy. To exploit the diverse functional abilities of NK cells for cancer immunotherapy, it is important to understand NK cell biology and the underlying regulatory mechanisms. The state of immune suppression prevalent in malignancies creates the need for innovative therapies. Oncolytic viruses are novel anticancer agents showing selective tropism for tumor cells and lacking pathogenicity in humans, but the use of oncolytic virotherapy (OVT) presents multiple challenges. An increasing body of evidence suggests that the host immune response may critically influence the outcome of OVT. Classically, the immune system is thought to limit the efficacy of therapy through virus clearance mediated by innate immune effectors or through adaptive antiviral immune responses eliminating infected cells. Effective strategies do need to be designed in OVT to circumvent the early antiviral activity of NK cells and to augment late NK-cell-mediated antitumor responses. The intrinsic immunostimulating capacity of oncolytic viruses and the possibility of engineering them to express heterologous immunostimulatory molecules (eg, cytokines) support the use of these agents to enhance antitumor immune responses besides inducing direct oncolytic effects. OVT has indeed shown promising therapeutic outcomes in various clinical trials. Here, we review the biology of NK cells, strategies involving NK cells for achieving cancer therapy, and, more particularly, the emerging role of NK cells in OVT.

NK-cell-dependent killing of colon carcinoma cells is mediated by natural cytotoxicity receptors (NCRs) and stimulated by parvovirus infection of target cells.

BACKGROUND: Investigating how the immune system functions during malignancies is crucial to developing novel therapeutic strategies. Natural killer (NK) cells, an important component of the innate immune system, play a vital role in immune defense against tumors and virus-infected cells. The poor survival rate in colon cancer makes it particularly important to develop novel therapeutic strategies. Oncolytic viruses, in addition to lysing tumor cells, may have the potential to augment antitumor immune responses. In the present study, we investigate the role of NK cells and how parvovirus H-1PV can modulate NK-cell mediated immune responses against colon carcinoma. METHODS: Human NK cells were isolated from the blood of healthy donors. The cytotoxicity and antibody-mediated inhibition of NK cells were measured in chromium release assays. Phenotypic assessment of colon cancer and dendritic cells was done by FACS. The statistical significance of the results was calculated with Student's t test (*p <0.05; **, p < 0.01; ***, p < 0.001). RESULTS: We show that IL-2-activated human NK cells can effectively kill colon carcinoma cells. Killing of colon carcinoma cells by NK cells was further enhanced upon infection of the former cells with parvovirus H-1PV. H-1PV has potent oncolytic activity against various tumors, yet its direct killing effect on colon carcinoma cells is limited. The cytotoxicity of NK cells towards colon carcinoma cells, both mock- and H-1PV-infected, was found to be mostly mediated by a combination of natural cytotoxicity receptors (NCRs), namely NKp30, 44, and 46. Colon carcinoma cells displayed low to moderate expression of NK cell ligands, and this expression was modulated upon H-1PV infection. Lysates of H-1PV-infected colon carcinoma cells were found to increase MHC class II expression on dendritic cells. CONCLUSIONS: Altogether, these data suggest that IL-2-activated NK cells actively kill colon carcinoma cells and that this killing is mediated by several natural cytotoxicity receptors (NCRs) in combination. Additionally, in association with parvovirus H-1PV, IL-2-activated NK cells have the potential to boost immune responses against colon cancer.

Antitumoral activity of parvovirus-mediated IL-2 and MCP-3/CCL7 delivery into human pancreatic cancer: implication of leucocyte recruitment.

Pancreatic ductal adenocarcinoma (PDAC) represents the fourth leading cause of cancer-related death in western countries. The patients are often diagnosed in advanced metastatic stages, and the prognosis remains extremely poor with an overall 5-year survival rate less than 5 %. Currently, novel therapeutic strategies are being pursued to combat PDAC, including oncolytic viruses, either in their natural forms or armed with immunostimulatory molecules. Natural killer cells are critical players against tumours and infected cells. Recently, we showed that IL-2-activated human NK cells displayed killing activity against PDAC cells, which could further be enhanced through the infection of PDAC cells with the rodent parvovirus H-1PV. In this study, the therapeutic efficacy of parvovirus-mediated delivery of three distinct cyto/chemokines (Il-2, MCP-3/CCL7 and IP-10/CXCL10) was evaluated in xenograft models of human PDAC. We show here that activated NK and monocytic cells were found to be recruited by PDAC tumours upon infection with parvoviruses armed with IL-2 or the chemokine MCP-3/CCL7, resulting in a strong anti-tumour response.

Enhancement of NK cell antitumor responses using an oncolytic parvovirus.

Natural killer (NK) cells play a vital role in the rejection of tumors. Pancreatic ductal adenocarcinoma (PDAC), however, remains a poor prognosis malignancy, due to its resistance to radio- and chemotherapy, and low immunogenicity. We demonstrate here that IL-2-activated human NK cells are able to kill PDAC cells. Currently, novel strategies are being pursued to combat PDAC. In this regard, oncolytic viruses, in addition to killing tumor cells, may also have the potential to augment antitumor immune responses. We found that, besides having an intrinsic oncolytic activity, parvovirus H-1PV is able to enhance NK cell-mediated killing of PDAC cells. Our results show that H-1PV infection of Panc-1 cells increases NK cell capacity to release IFN-γ, TNF-α and MIP-1α/ß. Multiple activating receptors are involved in the NK cell-mediated killing of Panc-1 cells. Indeed, blocking of the natural cytotoxicity receptors-NKp30, 44 and 46 in combination, and NKG2D and DNAM1 alone inhibit the killing of Panc-1 cells. Interestingly, H-1PV infection of Panc-1 cells overcomes the part of inhibitory effects suggesting that parvovirus may induce additional NK cell ligands on Panc-1 cells. The enhanced sensitivity of H-1PV-infected PDAC cells to NK cell-dependent killing could be traced back to the upregulation of the DNAM-1 ligand, CD155 and to the downregulation of MHC class I expression. Our data suggests that NK cells display antitumor potential against PDAC and that H-1PV-based oncolytic immunotherapy could further boost NK cell-mediated immune responses and help to develop a combinatorial therapeutic approach against PDAC.

Regulation of NK cell activity by 2B4, NTB-A and CRACC.

2B4, NTB-A and CRACC are members of the recently defined family of SLAM-related receptors. Here we review the role of these receptors for the regulation of Natural Killer cell function and describe the current knowledge about the signal transduction of these receptors. Finally, we critically analyze some controversial data about the function of 2B4 in mouse and man.

Serial killing of tumor cells by human natural killer cells--enhancement by therapeutic antibodies.

BACKGROUND: Natural killer cells are an important component of the innate immune system. Anti-cancer therapies utilizing monoclonal antibodies also rely on the cytotoxicity of NK cells for their effectiveness. Here, we study the dynamics of NK cell cytotoxicity. METHODOLOGY/PRINCIPAL FINDINGS: We observe that IL-2 activated human NK cells can serially hit multiple targets. Using functional assays, we demonstrate that on an average, a single IL-2 activated NK cell can kill four target cells. Data using live video microscopy suggest that an individual NK cell can make serial contacts with multiple targets and majority of contacts lead to lysis of target cells. Serial killing is associated with a loss of Perforin and Granzyme B content. A large majority of NK cells survive serial killing, and IL-2 can replenish their granular stock and restore the diminished cytotoxicity of 'exhausted' NK cells. IL-2 and IL-15 are equally effective in enhancing the killing frequency of resting NK cells. Significantly, Rituximab, a therapeutic monoclonal antibody increases the killing frequency of both resting and IL-2 activated NK cells. CONCLUSION/SIGNIFICANCE: Our data suggest that NK cell-based therapies for overcoming tumors rely on their serial killing ability. Therefore, strategies augmenting the killing ability of NK cells can boost the immune system and enhance the effectiveness of monoclonal antibody-based therapies.

Fine-tuning of immune responses by SLAM-related receptors.

The modulation of antigen receptor signals is important for a productive immune response. The main function of the recently identified members of the signaling lymphocyte activating molecule (SLAM)-related receptors (SRR) is the fine-tuning of immune cell activation. Disruption of SRR function is the cause for severe immune disorders such as X-linked lymphoproliferative syndrome (XLP), where XLP patients carry a mutation in SLAM-associated protein (SAP) (SH2D1A), an important adaptor molecule for the signal transduction of SRR. Recent data also suggest that SRR may play a role in autoimmune diseases and the function of hematopoietic stem and progenitor cells. Here, we review the current understanding of SRR function in different immune cells.

Coordinate expression of CC chemokine ligand 5, granulysin, and perforin in CD8+ T cells provides a host defense mechanism against Mycobacterium tuberculosis.

The ability of CD8+ T cells to kill intracellular pathogens depends upon their capacity to attract infected cells as well as their secretion of cytolytic and antimicrobial effector molecules. We examined the Ag-induced expression of three immune effector molecules contained within cytoplasmic granules of human CD8+ T cells: the chemokine CCL5, the cytolytic molecule perforin, and the antimicrobial protein granulysin. Macrophages infected with virulent Mycobacterium tuberculosis triggered the expression of CCL5 in CD8+ T cells only in donors with previous exposure to the tuberculosis bacteria, not in naive donors. Functionally, CCL5 efficiently attracted M. tuberculosis-infected macrophages, but failed to exert direct antibacterial activity. Infected macrophages also triggered the expression of granulysin in CD8+ T cells, and granulysin was found to be highly active against drug-susceptible and drug-resistant M. tuberculosis clinical isolates. The vast majority of CCL5-positive cells coexpressed granulysin and perforin. Taken together, this report provides evidence that a subset of CD8+ T cells coordinately expresses CCL5, perforin and granulysin, thereby providing a host mechanism to attract M. tuberculosis-infected macrophages and kill the intracellular pathogen.

Inverse correlation of maturity and antibacterial activity in human dendritic cells.

Dendritic cells (DCs) are a key part of host defense against microbial pathogens, being part of the innate immune system, but also instructing the adaptive T cell response. This study was designed to evaluate whether human DCs directly contribute to innate immunity by killing intracellular bacteria, using tuberculosis as a model. DCs were detected in bronchoalveolar lavage samples indicating that DCs are available for immediate interaction with Mycobacterium tuberculosis (M. Tb) after inhalation of the pathogen. The phenotype of DC in bronchoalveolar lavage closely resembles monocyte-derived immature DC (iDC) according to the expression of CD1a, CD83, and CCR7. The antimicrobial activity of iDC against intracellular M. Tb inversely correlated with TNF-alpha-release and was enhanced by treatment with anti-TNF-alpha Abs. Differentiation of iDC into mature DC by addition of TNF-alpha or activation via Toll-like receptors further reduced killing of M. Tb. The antibacterial activity against intracellular M. Tb of all DCs was significantly lower than alveolar macrophages. Therefore, the maintenance of a pool of DCs at the site of disease activity in tuberculosis, and the maturation of these DC by TNF-alpha provides a mechanism by which M. Tb escapes the innate immune system.