Characterization and immunogenicity assessment of MERS-CoV pre-fusion spike trimeric oligomers as vaccine immunogen.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a lethal beta-coronavirus that emerged in 2012. The virus is part of the WHO blueprint priority list with a concerning fatality rate of 35%. Scientific efforts are ongoing for the development of vaccines, anti-viral and biotherapeutics, which are majorly directed toward the structural spike protein. However, the ongoing effort is challenging due to conformational instability of the spike protein and the evasion strategy posed by the MERS-CoV. In this study, we have expressed and purified the MERS-CoV pre-fusion spike protein in the Expi293F mammalian expression system. The purified protein was extensively characterized for its biochemical and biophysical properties. Thermal stability analysis showed a melting temperature of 58°C and the protein resisted major structural changes at elevated temperature as revealed by fluorescence spectroscopy and circular dichroism. Immunological assessment of the MERS-CoV spike immunogen in BALB/c mice with AddaVaxTM and Imject alum adjuvants showed elicitation of high titer antibody responses but a more balanced Th1/Th2 response with AddaVaxTM squalene like adjuvant. Together, our results suggest the formation of higher-order trimeric pre-fusion MERS-CoV spike proteins, which were able to induce robust immune responses. The comprehensive characterization of MERS-CoV spike protein warrants a better understanding of MERS spike protein and future vaccine development efforts.

Novel educational training of para medical professionals in cervical cancer screening.

Background: Cervical cancer is a public health problem in India due to weak national screening policy compounded by lack of resources including scarcity of trained personnel to carry out community-based screening program. Para medical professionals (PMPs) are closely related to women in local communities. Hence, training PMPs by incorporating novel technology and reduced time duration to achieve adequate competence in screening is an area underutilized and needs to be explored. Materials and methods: A pilot cross sectional analytical study was conducted at a tertiary referral cancer center using a shorter version of educational intervention of 2 weeks duration (EI2W) involving PMPs. Pre- and post-training assessment of knowledge, attitude, and practice (KAP) was done using questionnaires consisting of 5 domains viz. awareness of cervical cancer, awareness of cervical pre-cancer, practical screening methodology (practice oriented), data management and aspects of human papilloma virus (HPV). Wilcoxon signed-rank test was used for comparison and the degree of change was measured using analysis of covariance (ANCOVA). A p value of <0.05 was considered significant. Results: 118 PMPs were included. There was a significant improvement in scores of all domains (except cervical pre-cancer domain), following introduction of EI2W. Knowledge scores, post EI2W was better in Auxiliary Nurse Midwives (ANMs) than other participants. Awareness regarding cervical cancer was higher with more years of experience. The KAP analysis showed excellent interrater reliability in the practice 0.726 (0.649-0.792) followed by knowledge domain 0.711 (0.626-0.783). Conclusion: EI2W was effective in significantly improving the competence of PMPs, thus reducing human resource constraints in cervical cancer prevention and elimination.

Intradermal Immunization of Soluble Influenza HA Derived from a Lethal Virus Induces High Magnitude and Breadth of Antibody Responses and Provides Complete Protection In Vivo.

Immunogens mimicking the native-like structure of surface-exposed viral antigens are considered promising vaccine candidates. Influenza viruses are important zoonotic respiratory viruses with high pandemic potential. Recombinant soluble hemagglutinin (HA) glycoprotein-based protein subunit vaccines against Influenza have been shown to induce protective efficacy when administered intramuscularly. Here, we have expressed a recombinant soluble trimeric HA protein in Expi 293F cells and purified the protein derived from the Inf A/Guangdong-Maonan/ SWL1536/2019 virus which was found to be highly virulent in the mouse. The trimeric HA protein was found to be in the oligomeric state, highly stable, and the efficacy study in the BALB/c mouse challenge model through intradermal immunization with the prime-boost regimen conferred complete protection against a high lethal dose of homologous and mouse-adapted InfA/PR8 virus challenge. Furthermore, the immunogen induced high hemagglutinin inhibition (HI) titers and showed cross-protection against other Inf A and Inf B subtypes. The results are promising and warrant trimeric HA as a suitable vaccine candidate.

Biophysical and Biochemical Characterization of the Receptor Binding Domain of SARS-CoV-2 Variants.

The newly emerging SARS-CoV-2 variants are potential threat and posing new challenges for medical intervention due to high transmissibility and escaping neutralizing antibody (NAb) responses. Many of these variants have mutations in the receptor binding domain (RBD) of SARS-CoV-2 spike protein that interacts with the host cell receptor. Rapid mutation in the RBD through natural selection to improve affinity for host receptor and antibody pressure from vaccinated or infected individual will greatly impact the presently adopted strategies for developing interventions. Understanding the nature of mutations and how they impact the biophysical, biochemical and immunological properties of the RBD will help immensely to improve the intervention strategies. To understand the impact of mutation on the protease sensitivity, thermal stability, affinity for the receptor and immune response, we prepared several mutants of soluble RBD that belong to the variants of concern (VoCs) and interest (VoIs) and characterize them. Our results show that the mutations do not impact the overall structure of the RBD. However, the mutants showed increase in the thermal melting point, few mutants were more sensitive to protease degradation, most of them have enhanced affinity for ACE2 and some of them induced better immune response compared to the parental RBD.

Designing and characterization of a SARS-CoV-2 immunogen with receptor binding motif grafted on a protein scaffold: An epitope-focused vaccine approach.

The COVID-19 pandemic caused by SARS-CoV-2 has a significant burden on the economy and healthcare around the world. Vaccines are the most effective tools to fight infectious diseases by containing the spread of the disease. The current vaccines against SARS-CoV-2 are mostly based on the spike protein of SARS-CoV-2, which is large and has many immune-dominant non-neutralizing epitopes that may effectively skew the antibody response towards non-neutralizing antibodies. Here, we have explored the possibility of immune-focusing the receptor binding motif (RBM) of the spike protein of SARS-CoV-2 that induces mostly neutralizing antibodies in natural infection or in vacinees. The result shows that the scaffolded RBM can bind to Angiotensin Converting Enzyme 2 (ACE2) although with low affinity and induces a strong antibody response in mice. The immunized sera can bind both, the receptor binding domain (RBD) and the spike protein, which holds the RBM in its natural context. Sera from the immunized mice showed robust interferon γ response but poor neutralization of SARS-CoV-2 suggesting presence of a predominant T cell epitope on scaffolded RBM. Together, we provide a strategy for inducing strong antigenic T cell response which could be exploited further for future vaccine designing and development against SARS-CoV-2 infection.

Anti-mycobacterial activity evaluation of designed peptides: cryptic and database filtering based approach.

Worldwide, TB is one of the deadly airborne diseases, which accounts for 10.4 million deaths annually. Serious toxicity issue, prolonged treatment regimens of the current drugs, rise in multidrug-resistant strains, and the unique defensive mechanism makes the development of novel therapeutic molecules against Mycobacterium tuberculosis (MT) an urgent need. As MT has a lengthy latent phase and unique cell wall architecture, a reasonable approach is needed to find molecules having a different killing mechanism rather than traditional approaches. Host defence peptides (HDPs) will be the most promising alternative, potential therapeutic candidates as they target the microbial membrane in particular and are an essential part of the innate immunity of humans. This works demonstrates the utility of "Database filtering" and three-dimensional (3D) modelling approach in finding novel AMPs with appreciable activity towards MT. Results of this study indicate that peptides with 70% hydrophobicity, but without hydrophobicity patches (> 4 hydrophobic amino acids in series) and charge of + 4 or + 5 are most likely to be good anti-tubercular candidates.

1,2,3-triazole-thiazole hybrids: Synthesis, in vitro antimicrobial activity and antibiofilm studies.

A new series of triazole-thiazole hybrids were designed, synthesized by the Multi-component reaction approach and evaluated in vitro antimicrobial activity. Most of the tested series of compounds exhibited promising inhibitory activity against the bacterial strains with values in the range of 2.8 to 15.7 µM. The compounds 8i-8l and 8r showed potential-Candida activity against various Candida strains with spectrum values in the range 5.9-14.2 µM. Further, anti-biofilm and toxicity profiles for the potent compounds were also tested, and it was observed that the compounds 8i, 8k, and 8l were found to inhibit the biofilm formation with IC50 values of 6.6, 16.6 and 15.9 µM, respectively against Bacillus subtilis MTCC 121. Besides, 8k and 8l also displayed promising biofilm formation inhibitory activity towards Staphylococcus aureus MTCC 96 with IC50 values of 13.5 and 12.0 µM respectively. In summary, the activity results has emphasized the compounds 8k and 8l as potential leads for further development of antibacterial, anti-Candida, and anti-biofilm agents.

Synthesis and antimicrobial evaluation of piperic acid amides and their lower homologues.

Seven piperic acid amides along with their lower homologs (12) were synthesized using HATU-DIPEA coupling reagent. All the synthesized derivatives were evaluated for their antibacterial activities against Staphylococcus aureus, Pseudomonas aeruginosa, and vancomycin-resistant P. aeruginosa. They were found to be more active on P. aeruginosa than on S. aureus. However, they did not exhibit potent activity on Vancomycin resistant P. aeruginosa. Among the tested compounds, methylenedioxycinnamic acid amide of anthranilic acid (MDCA-AA, 2a) was found to be most active against S. aureus with MIC of 3.125 µg/ml. The PAS and INH amides of piperic acid were screened against Mycobacterium tuberculosis H37Ra strain. They were found to be most active among all the tested compounds but were found to be less active than the standard drug, isoniazid.

In-house chemical library repurposing: A case example for Pseudomonas aeruginosa antibiofilm activity and quorum sensing inhibition.

Hit, Lead & Candidate Discovery Drug repurposing has become a recent trend in drug development programs, where previously developed drugs are explored for hit and redeveloped into potential therapeutic agents for new diseases. Globally, in any drug development program, a series of molecules are synthesized and evaluated for the hypothesized activity. Hits are developed into lead molecules or drugs, whereas the negative ones are shelved in the lab with no immediate use. We in this project took the previously sidelined small chemical molecules to the next level of utility, where previously developed in-house small molecules library are tested for the unexplored biological relevant activity. As biofilm formation and quorum sensing play a vital role in bacterial pathogenesis, we believe that they could be one of the most effective targets for antimicrobial agents. In this study, we report the evaluation of 50 different compounds for anti-biofilm and anti-quorum sensing activity against Pseudomonas aeruginosa. Out of the screened compounds, three hydrazine-carboxamide hybrid derivatives showed promising anti-biofilm property and inhibition of pyocyanin production without any direct antimicrobial activity and cytotoxicity issues. Hydrazine-carboxamide hybrids can be a new class and promising leads for further anti-biofilm and anti-virulence development against microbial infections.

Coumarin-benzimidazole hybrids as a potent antimicrobial agent: synthesis and biological elevation.

Molecular hybridization approach is an emerging tool in drug discovery for designing new pharmacophores with biological activity. A novel, new series of coumarin-benzimidazole hybrids were designed, synthesized and evaluated for their broad spectrum antimicrobial activity. Among all the synthesized molecules, compound (E)-3-(2-1H-benzo[d]imidazol-1-yl)-1-((4-chlorobenzyl)oxy)imino)ethyl)-2H-chromen-2-one showed the most promising broad spectrum antibacterial activity against Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis and Proteus vulgaris. In addition, it has showed no cytotoxicity and hemolysis at 10 times the MIC concentration. SAR studies indicate that position of the chlorine atom in the hybrid critically determines the antibacterial activity.