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Vaccines as a remedy for antimicrobial resistant bacterial infections

Periodic Reporting for period 2 - vAMRes (Vaccines as a remedy for antimicrobial resistant bacterial infections)

Okres sprawozdawczy: 2020-05-01 do 2021-10-31

Anti-microbial resistance (AMR) represents one of the most challenging public health issues of our times. The World Health Organization (WHO) estimated that more than 10 million people will lose their lives to AMR each year by 2050. Urgent measures are therefore needed in order to tackle this problem and find innovative solutions that help diagnosis and treatment. In addition, the recent pandemic caused by SARS-CoV-2 required discovery and development of medicines that could contain viral spread and accelerate recovery of infected patients.
In this context, the ERC Advanced Grant vAMRes focuses on the discovery of monoclonal antibodies (mAbs) against anti-microbial resistant bacteria (Neisseria gonorrhoeae and Escherichia coli) and against the SARS-CoV-2 virus for therapy and for supporting rational vaccine design. During the reporting period, research activities have been conducted on mAbs against N. gonorrhoeae and against SARS-CoV-2.
Neisseria gonorrhoeae: a clinical protocol approved by the ethics committee at the University Hospital in Siena allowed the recruitment of volunteers who underwent vaccination with the anti-meningococcus vaccine Bexsero. The rationale for this approach relies on observations published retrospectively in Petousis-Harris et al., 2017, where reduced cases of gonorrhoea were reported in populations vaccinated with Bexsero. Thousands of Plasmacells (PCs) and Memory B Cells (MBCs) were isolated from the blood of the vaccinated people by means of single-cell sorting. Subsequent Enzyme-linked Immunosorbent Assay (ELISA) and Luminex-based screening procedures identified a few hundred mAbs from PCs and from MBCs capable of binding multiple N. gonorrhoeae strains with different specificities. Conventional functional assays that assessed the potency of the mAbs against the pathogen, thus causing bacterial death, were applied alongside new, innovative assays based on the high-content confocal microscopy platform Opera Phenix. Results demonstrated bactericidal activity for several N. gonorrhoeae-specific mAbs whose target is now being identified. Moreover, thanks to the engineering of N. gonorrhoeae strains to express fluorescent proteins, adhesion and infection assays using specific cell lines were optimized by confocal microscopy. These assays will be exploited for testing mAbs and evaluating their activity in infection settings.
SARS-CoV-2: By single cell sorting 4,277 SARS-CoV-2 spike protein specific MBCs from 14 Covid-19 survivors, 453 neutralizing antibodies were identified. Only 1.4% of them neutralized the authentic virus in vitro with a potency of 1-10 ng/mL. The most potent neutralizing antibodies recognized the Receptor Binding Domain (RBD), followed by antibodies that recognize the S1 domain, the S-protein trimer and the S2 subunit. The three most potent mAbs, engineered to reduce the risk of antibody dependent enhancement of disease (ADE), prolong half-life and increase tissue distribution, neutralized the authentic virus with less than 10 ng/mL and protected hamsters from viral challenge at a concentration as low as 0.25 mg/Kg (Andreano et al., 2021). Importantly, the most potent mAb (named MAD0004J08) was shown to be active against the so-called Alpha, Beta, Gamma and Delta variants of SARS-CoV-2. MAD0004J08 has been produced on the large scale by an Italian pharmaceutical company and is now in clinical trials. Phase I was recently successfully completed and phase II has started with the administration of the candidate mAb to infected patients by intramuscular injection.
Thanks to the availability of the most recent technologies, relevant progress has been made in the mAb discovery process. This resulted in the identification of the mAb candidate MAD0004J08, which is now in Phase II clinical trials. This mAb is characterized by unprecedented potency, which allows intramuscular administration, and by the ability to neutralize all of the SARS-CoV-2 variants identified so far. If positive, Phase II results will support the approval for emergency use in COVID-19 positive patients. Additional work is now being performed to identify other equally potent mAbs that could be combined with MAD0004J08.

Within the N. gonorrhoeae project, most of the progress has been made in the development of confocal microscopy-based assays, which will soon replace conventional, low-throughput assays, thus bringing microbiology into the 21st century. Data generated so far will be further consolidated and enriched by in vivo experiments which will validate mAb potency in infection settings.
Monoclonal Antibodies Isolation process