key: cord-0826235-utqfe26r
authors: Capone, Stefania; Raggioli, Angelo; Gentile, Michela; Battella, Simone; Lahm, Armin; Sommella, Andrea; Contino, Alessandra Maria; Urbanowicz, Richard A.; Scala, Romina; Barra, Federica; Leuzzi, Adriano; Lilli, Eleonora; Miselli, Giuseppina; Noto, Alessia; Ferraiuolo, Maria; Talotta, Francesco; Tsoleridis, Theocharis; Castilletti, Concetta; Matusali, Giulia; Colavita, Francesca; Lapa, Daniele; Meschi, Silvia; Capobianchi, Maria; Soriani, Marco; Folgori, Antonella; Ball, Jonathan K.; Colloca, Stefano; Vitelli, Alessandra
title: Immunogenicity of a new gorilla adenovirus vaccine candidate for COVID-19
date: 2021-04-23
journal: Mol Ther
DOI: 10.1016/j.ymthe.2021.04.022
sha: 41b2b31a1723fb1466c2cf434b0a64e43b9d0fe3
doc_id: 826235
cord_uid: utqfe26r

The COVID-19 pandemic caused by the emergent SARS-CoV-2 coronavirus threatens global public health and there is an urgent need to develop safe and effective vaccines. Here we report the generation and the preclinical evaluation of a novel replication-defective gorilla adenovirus-vectored vaccine encoding the pre-fusion stabilized Spike (S) protein of SARS-CoV2. We show that our vaccine candidate, GRAd-COV2, is highly immunogenic both in mice and macaques, eliciting both functional antibodies which neutralize SARS-CoV-2 infection and block Spike protein binding to the ACE2 receptor, and a robust, Th1-dominated cellular response. We show here that the pre-fusion stabilized Spike antigen is superior to the wild type in inducing ACE2-interfering, SARS-CoV2 neutralizing antibodies. To face the unprecedented need for vaccine manufacturing at massive scale, different GRAd genome deletions were compared to select the vector backbone showing the highest productivity in stirred tank bioreactors. This preliminary dataset identified GRAd-COV2 as a potential COVID-19 vaccine candidate, supporting the translation of GRAd-COV2 vaccine in a currently ongoing Phase I clinical trial (NCT04528641).

FACS analysis revealed a 10% mean frequency of IFN-γ secreting, Spike-specific CD8+ T cells, 162 and a lower but clearly measurable Th1 dominated CD4+ T cell response ( Fig 5B) . Type 2 163 cytokines IL-4 and IL-13 and IL-17 secretion were also investigated by ICS and ELISpot, but 164 were undetectable or below the limit of detection of our assays. 165 To benchmark the potency of the novel GRAd32 vector in comparison with other described 166 simian adenoviral vectors 4 , we have performed a dose-response immunization exploring a range 167 of doses from 1x10 9 to 1x10 5 vp measuring IFN-γ secreting cells by ELISpot in the spleen 3 168 weeks after vaccination ( Fig S5) . Detectable immune responses in all animals were already 169 observed at the dose 1x10 6 vp, revealing the strong immunological potency of the new gorilla 170 vector. As a control for assay specificity, BALB/c mice were immunized with a GRAd32 vector 171 encoding the HIV-1 gag antigen. No humoral or cellular responses to the Spike protein or to the and T cell responses to gag were measured ( Fig S6) .

were taken five weeks after immunization with a single administration at 1x10 9 vp of GRAd32 176 vectors encoding the wt S or S-2P. We observed similar induction of total anti-S IgG in spite of 177 significantly higher neutralization titers for the S-2P antigen ( Fig 6A) higher than those measured in serum obtained from COVID-19 convalescent patients, using the 199 same MN assay ( Fig 7E) .

PBMCs were collected 2 and 8 weeks after immunization and T cell response were measured by 201 IFN-γ and IL4 ELISpot assay ( Fig 8A) . Strong IFN-γ secreting T cells responses were detected 202 in all animals at week 2 (700 -3500 SFC/10 6 PBMCs), which somewhat contracted at week 8 203 but remained at high level (400 -2500 SFC/10 6 PBMCs). Instead, IL-4 levels were too low to be 204 detected in macaques by ELISpot analysis (Fig 8A) . The animals immunized with S-2P showed 205 the highest number of IFN-γ secreting T cells (Fig 8B) , and S1 region of the Spike protein was 

In this report we describe the development of a COVID-19 vaccine candidate based on a novel 214 simian adenovirus, GRAd32, isolated from a captive gorilla and belonging to species C 215 adenoviruses. We show here that a single immunization with the GRAd-COV2 vector encoding a well were seeded in 96 well plates the day before the assay. Each adenoviral vector (Ad5 or

Pseudotyped viruses were produced as previously described 25 . Briefly, 1.5×10 6 HEK293T cells 509 were seeded overnight in a 10 cm diameter Primaria-coated dish (Corning, Flintshire, UK). VeroE6 cells/well were plated in white 96-well tissue culture plates (Corning) and incubated 520 overnight at 37°C. The following day, SARS-CoV-2 pseudotypes were incubated with heat-521 inactivated sera for 1 hr at RT before being added to cells for 4 h. Following this, sera and media 522 were discarded, and 200 µl DMEM was added to the cells. After 72 h, media was discarded, and

The study was performed in the framework of the special project "vaccine against SARS-CoV- , et al. (2004) . Quantitative adenovirus Disease. medRxiv, 2020 .2008 .2022 .20176834. 10.1101 /2020 

A Review of SARS-CoV-2 and the Ongoing Clinical Trials

Chimpanzee 604 adenoviral vectors as vaccines -challenges to move the technology into the fast lane

Chimpanzee adenovirus-and MVA-608 vectored respiratory syncytial virus vaccine is safe and immunogenic in adults

Vaccine vectors derived from a large 612 collection of simian adenoviruses induce potent cellular immunity across multiple species

Use of ChAd3-EBO-Z Ebola virus vaccine 619 in Malian and US adults, and boosting of Malian adults with MVA-BN-Filo: a phase 1, single-620 blind, randomised trial, a phase 1b, open-label and double-blind, dose-escalation trial, and a 621 nested, randomised, double-blind, placebo-controlled trial

Diagnosing 625 collisions of magnetized, high energy density plasma flows using a combination of collective

Thomson scattering, Faraday rotation, and interferometry (invited)

Safety and immunogenicity of the

ChAdOx1 nCoV-19 vaccine against SARS-CoV-2: a preliminary report of a phase 1/2, single-blind, 631 randomised controlled trial

633 epidemiologic studies and in the design of adenovector vaccines

Efficacy assessment of a cell-mediated 643 immunity HIV-1 vaccine (the Step Study): a double-blind, randomised, placebo-controlled, test-644 of-concept trial

A gene transfer vector-646 cell line system for complete functional complementation of adenovirus early regions E1 and E4

Structure-based design of a fusion glycoprotein 650 vaccine for respiratory syncytial virus

Structure-based design of a 653 quadrivalent fusion glycoprotein vaccine for human parainfluenza virus types 1-4

An antibody against the F glycoprotein inhibits Nipah 657 and Hendra virus infections

Structure and immune recognition of 660 trimeric pre-fusion HIV-1 Env

Single-shot Ad26 vaccine protects against

SARS-CoV-2 in rhesus macaques

Antigen expression determines adenoviral 666 vaccine potency independent of IFN and STING signaling

Trial of Ad26.COV2.S Covid-19 Vaccine

Rapid COVID-19 vaccine development

Interactive Tree Of Life (iTOL) v4: recent updates and new 677 developments

MUSCLE: multiple sequence alignment with high accuracy and high 679 throughput

Pseudotype Neutralization Assays: From Laboratory 681 Bench to Data Analysis