A Structural Landscape of Neutralizing Antibodies Against SARS-CoV-2 Receptor Binding Domain.

SARS-CoV-2, the novel coronavirus responsible for the ongoing COVID-19 pandemic, has been spreading rampantly. The global scientific community has responded rapidly to understand immune correlates of protection to develop vaccines and immunotherapeutics against the virus. The major goal of this mini review is to summarize current understanding of the structural landscape of neutralizing antibodies (nAbs) that target the receptor binding domain (RBD) of viral spike (S) glycoprotein. The RBD plays a critical role in the very first step of the virus life cycle. Better understanding of where and how nAbs bind the RBD should enable identification of sites of vulnerability and facilitate better vaccine design and formulation of immunotherapeutics. Towards this goal, we compiled 38 RBD-binding nAbs with known structures. Review of these nAb structures showed that (1) nAbs can be divided into five general clusters, (2) there are distinct non-neutralizing faces on the RBD, and (3) maximum of potentially four nAbs could bind the RBD simultaneously. Since most of these nAbs were isolated from virus-infected patients, additional analyses of vaccine-induced nAbs could facilitate development of improved vaccines.

Induction of Potent and Durable Neutralizing Antibodies Against SARS-CoV-2 Using a Receptor Binding Domain-Based Immunogen.

A novel betacoronavirus (SARS-CoV-2) that causes severe pneumonia emerged through zoonosis in late 2019. The disease, referred to as COVID-19, has an alarming mortality rate and it is having a devastating effect on the global economy and public health systems. A safe, effective vaccine is urgently needed to halt this pandemic. In this study, immunogenicity of the receptor binding domain (RBD) of spike (S) glycoprotein was examined in mice. Animals were immunized with recombinant RBD antigen intraperitoneally using three different adjuvants (Zn-chitosan, Alhydrogel, and Adju-Phos), and antibody responses were followed for over 5 months. Results showed that potent neutralizing antibodies (nAbs) can be induced with 70% neutralization titer (NT70) of ~14,580 against live, infectious viruses. Although antigen-binding antibody titers decreased gradually over time, sufficiently protective levels of nAbs persisted (NT80 >2,430) over the 5-month observation period. Results also showed that adjuvants have profound effects on kinetics of nAb induction, total antibody titers, antibody avidity, antibody longevity, and B-cell epitopes targeted by the immune system. In conclusion, a recombinant subunit protein immunogen based on the RBD is a highly promising vaccine candidate. Continued evaluation of RBD immunogenicity using different adjuvants and vaccine regimens could further improve vaccine efficacy.

Rhinovirus-induces progression of lung disease in a mouse model of COPD via IL-33/ST2 signaling axis.

Rhinovirus (RV), which is associated with acute exacerbations, also causes persistent lung inflammation in patients with chronic obstructive pulmonary disease (COPD), but the underlying mechanisms are not well-known. Recently, we demonstrated that RV causes persistent lung inflammation with accumulation of a subset of macrophages (CD11b+/CD11c+), and CD8+ T cells, and progression of emphysema. In the present study, we examined the mechanisms underlying the RV-induced persistent inflammation and progression of emphysema in mice with COPD phenotype. Our results demonstrate that at 14 days post-RV infection, in addition to sustained increase in CCL3, CXCL-10 and IFN-γ expression as previously observed, levels of interleukin-33 (IL-33), a ligand for ST2 receptor, and matrix metalloproteinase (MMP)12 are also elevated in mice with COPD phenotype, but not in normal mice. Further, MMP12 was primarily expressed in CD11b+/CD11c+ macrophages. Neutralization of ST2, reduced the expression of CXCL-10 and IFN-γ and attenuated accumulation of CD11b+/CD11c+ macrophages, neutrophils and CD8+ T cells in COPD mice. Neutralization of IFN-γ, or ST2 attenuated MMP12 expression and prevented progression of emphysema in these mice. Taken together, our results indicate that RV may stimulate expression of CXCL-10 and IFN-γ via activation of ST2/IL-33 signaling axis, which in turn promote accumulation of CD11b+/CD11c+ macrophages and CD8+ T cells. Furthermore, RV-induced IFN-γ stimulates MMP12 expression particularly in CD11b+/CD11c+ macrophages, which may degrade alveolar walls thus leading to progression of emphysema in these mice. In conclusion, our data suggest an important role for ST2/IL-33 signaling axis in RV-induced pathological changes in COPD mice.

Quercetin prevents rhinovirus-induced progression of lung disease in mice with COPD phenotype.

Acute exacerbations are the major cause of morbidity and mortality in patients with chronic obstructive pulmonary disease (COPD). Rhinovirus, which causes acute exacerbations may also accelerate progression of lung disease in these patients. Current therapies reduces the respiratory symptoms and does not treat the root cause of exacerbations effectively. We hypothesized that quercetin, a potent antioxidant and anti-inflammatory agent with antiviral properties may be useful in treating rhinovirus-induced changes in COPD. Mice with COPD phenotype maintained on control or quercetin diet and normal mice were infected with sham or rhinovirus, and after 14 days mice were examined for changes in lung mechanics and lung inflammation. Rhinovirus-infected normal mice showed no changes in lung mechanics or histology. In contrast, rhinovirus-infected mice with COPD phenotype showed reduction in elastic recoiling and increase in lung inflammation, goblet cell metaplasia, and airways cholinergic responsiveness compared to sham-infected mice. Interestingly, rhinovirus-infected mice with COPD phenotype also showed accumulation of neutrophils, CD11b+/CD11c+ macrophages and CD8+ T cells in the lungs. Quercetin supplementation attenuated rhinovirus-induced all the pathologic changes in mice with COPD phenotype. Together these results indicate that quercetin effectively mitigates rhinovirus-induced progression of lung disease in a mouse model of COPD. Therefore, quercetin may be beneficial in the treatment of rhinovirus-associated exacerbations and preventing progression of lung disease in COPD.

Identification of dominant antibody-dependent cell-mediated cytotoxicity epitopes on the hemagglutinin antigen of pandemic H1N1 influenza virus.

Antibody-dependent cell-mediated cytotoxicity (ADCC) bridges innate and adaptive immunity, and it involves both humoral and cellular immune responses. ADCC has been found to be a main route of immune protection against viral infections in vivo. Hemagglutinin (HA) of influenza virus is highly immunogenic and considered the most important target for immune protection. Several potent cross-reactive HA-specific neutralizing monoclonal antibodies (MAbs) have been reported, and their conserved neutralizing epitopes have been revealed, but there has been no report so far about ADCC epitopes on HA. Here we identified two dominant ADCC epitopes, designated E1 (amino acids [aa] 92 to 117) and E2 (aa 124 to 159), on HA of pandemic H1N1 influenza virus by epitope mapping of convalescent-phase plasma IgG antibodies from six H1N1-infected human subjects in China that exhibited different levels of ADCC activity. The E1 and E2 ADCC epitopes overlapped with immunodominant epitopes of HA. Depletion of purified patient plasma IgG antibodies with EBY100 yeast cells expressing E1 or E2 decreased the ADCC activity of the IgG antibodies. E1 and E2 sequences were found to be highly conserved in H1N1 strains but less so in other subtypes of influenza A viruses. Our study may aid in designing immunogens that can elicit antibodies with high ADCC activity. Vaccine immunogens designed to include the structural determinants of potent broadly neutralizing antibodies and ADCC epitopes may confer comprehensive immune protection against influenza virus infection.

Resolution of immune response by recombinant transforming growth factor-beta (rTGF-ß) during influenza A virus infection.

BACKGROUND & OBJECTIVES: Replication of influenza A virus in the respiratory tract leads to cell damage and liberation of cytokines and chemokines. The in vivo cytokine induction and modulation by recombinant transforming growth factor- ß1 (rTGF-ß1) has not been studied. Therefore, in the present study the effect of rTGF-ß1, a potent immunomodulatory cytokine which has anti-inflammatory properties and downregulates the release of inflammatory molecules, against influenza-virus infection in the airway of mice was investigated. METHODS: rTGF-ß1 was administered intravenously to mice with concomitant intranasal infection of influenza A/Udorn/317/72 (H3N2) virus, and the survival rate, virus titre, histopathological changes and levels of factors regulating inflammation in the airway fluid were analysed. RESULT: The immune response to influenza A virus was characterized by an influx of both macrophages and lymphocytes into the lungs of the infected host. rTGF-ß1 significantly suppressed virus multiplication and improved the survival rate of mice. rTGF-ß1 downregulated infiltration of neutrophils and the release of inflammatory molecules, such as interferon-gamma (IFN-γ), interleukin-1 ß (IL-1ß) and stimulated release of IL-10 that potentiates anti-inflammatory response into airway. INTERPRETATION & CONCLUSIONS: A generalized pulmonary inflammation does not contribute to viral clearance but represents an immunological background within which antiviral immunity operates. Treatment with rTGF-ß1 reduced macrophage count and neutrophils influx in lungs of infected mice.

Influenza a virus induced apoptosis: inhibition of DNA laddering & caspase-3 activity by zinc supplementation in cultured HeLa cells.

BACKGROUND & OBJECTIVE: The pathogenesis of influenza virus infection involves virus replication in epithelial cells of the respiratory tract and the consequent degeneration of infected cells. Influenza virus induces cellular degeneration following infection of cultured cells in vitro, and the cytopathic effect (CPE) occurs principally through apoptotic cell death. This study was undertaken to fi nd out the effect of zinc on influenza virus induced apoptosis in cultured HeLa cells. METHODS: The sub-confluent monolayer HeLa cells were used to study the effect of zinc on influenza virus induced apoptosis. The apoptotic markers viz., caspase-3 activity, phagocytic index, morphological changes, and DNA fragmentation were assayed. RESULTS: When HeLa cells were infected with a cell adapted pathogenic strain of influenza A (A/Udorn/ 317/72H(3)N(2)) virus, DNA fragmentation was observed in virus infected cells by 24 h post infection and caspase-3 activity was maximum at 4 h post infection after which it reached to plateau. Treatment of cells with 0.1 5mM concentration of zinc till 8 h post infection inhibited DNA fragmentation and also caspase 3 activity was decreased significantly up to 2 h post infection. INTERPRETATION & CONCLUSION: When the infected HeLa cells were incubated with adherent macrophages, efficient phagocytosis occurred and the release of virus into the culture medium was inhibited. These results suggested that inhibitory effect on influenza virus induced apoptotic death of cultured cells can be determined at an early stage of the infection by treatment of zinc.

Management of three-dimensional intrafraction motion through real-time DMLC tracking.

Tumor tracking using a dynamic multileaf collimator (DMLC) represents a promising approach for intrafraction motion management in thoracic and abdominal cancer radiotherapy. In this work, we develop, empirically demonstrate, and characterize a novel 3D tracking algorithm for real-time, conformal, intensity modulated radiotherapy (IMRT) and volumetric modulated arc therapy (VMAT)-based radiation delivery to targets moving in three dimensions. The algorithm obtains real-time information of target location from an independent position monitoring system and dynamically calculates MLC leaf positions to account for changes in target position. Initial studies were performed to evaluate the geometric accuracy of DMLC tracking of 3D target motion. In addition, dosimetric studies were performed on a clinical linac to evaluate the impact of real-time DMLC tracking for conformal, step-and-shoot (S-IMRT), dynamic (D-IMRT), and VMAT deliveries to a moving target. The efficiency of conformal and IMRT delivery in the presence of tracking was determined. Results show that submillimeter geometric accuracy in all three dimensions is achievable with DMLC tracking. Significant dosimetric improvements were observed in the presence of tracking for conformal and IMRT deliveries to moving targets. A gamma index evaluation with a 3%-3 mm criterion showed that deliveries without DMLC tracking exhibit between 1.7 (S-IMRT) and 4.8 (D-IMRT) times more dose points that fail the evaluation compared to corresponding deliveries with tracking. The efficiency of IMRT delivery, as measured in the lab, was observed to be significantly lower in case of tracking target motion perpendicular to MLC leaf travel compared to motion parallel to leaf travel. Nevertheless, these early results indicate that accurate, real-time DMLC tracking of 3D tumor motion is feasible and can potentially result in significant geometric and dosimetric advantages leading to more effective management of intrafraction motion.

Biological and epidemiological aspects of influenza virus H5N1 in context of India.

Since 1997, highly pathogenic avian influenza (HPAI) H5N1 virus crossed the species barriers from birds to humans and caused fatal disease, leading to great speculation about a possible influenza pandemic. This subtype is characterized by its pathogenicity in a large number of animal species and resistance to older class of antiviral drugs. At present, two out of three general conditions for the onset of pandemic have been met, emergence of new virus; and its ability to replicate in humans causing serious illness. Next influenza pandemic might be due to human to human transmission. This review addresses the biological and epidemiological aspects of influenza in context of India.

Effect of quercetin supplementation on lung antioxidants after experimental influenza virus infection.

In the mice, instillation of influenza virus A/Udorn/317/72(H3N2) intranasally resulted in a significant decrease in the pulmonary concentrations of catalase, reduced glutathione, and superoxide dismutase. There was a decrease in vitamin E level also. These effects were observed on the 5th day after viral instillation. Oral supplementation with quercetin simultaneous with viral instillation produced significant increases in the pulmonary concentrations of catalase, reduced glutathione, and superoxide dismutase. However, quercetin did not reverse the fall in vitamin E level associated with the viral infection. It is concluded that during influenza virus infection, there is "oxidative stress." Because quercetin restored the concentrations of many antioxidants, it is proposed that it may be useful as a drug in protecting the lung from the deleterious effects of oxygen derived free radicals released during influenza virus infection.