This is a Phase 1, open-label, non-randomized, dose-escalation study using three doses and two schemes of administration of a recombinant vaccine against SARS-CoV-2 based on a viral vector (Newcastle Disease virus) in 90 healthy volunteers at a single research site in Mexico City.

The lack of highly effective treatments against COVID-19 and the social and economic impact that the current pandemic has exerted on public health highlights the uncontested importance of developing vaccines that, in addition to their safety and ability to induce a protective response, are logistically suitable for massive administration across a variety of countries and settings.

This is the first clinical study of the development program of a vaccine based on a unique recombinant viral vector technology that has been successful in the design of avian vaccines and that has no contraindication for use in humans.

The recombinant vaccine subject to research in this study is based on an active viral vector of a recombinant Newcastle Disease virus (rNDV) LaSota strain, in which the gene that codes for the S glycoprotein of SARS-CoV-2 has been inserted.

The Newcastle Disease Virus (NDV) is a paramyxovirus responsible for the Newcastle Disease in birds. There are three main families of NDV according to the level of virulence. The one with the lowest virulence is the lentogenic group. One lentogenic viral strain is LaSota (NDV_LS), which is broadly used in the development of avian vaccines. The LaSota strain seems to replicate only at the site of inoculation and, although it does not reach the lymph nodes, it reduces the induction of pro-inflammatory cytokines while boosting a robust protective immune response. Very importantly, this virus cannot insert itself into the human genome.

One of the key factors for an increased virulence in NDV is the activation of the cleavage site that corresponds to the protein F precursor phenotype. In highly virulent strains, the cleavage is performed by ubiquitous intracellular proteins, which leads to a widespread replication in birds.

However, the cleavage site in attenuated or non-virulent strains is activated by a secretory protease which restrains viral replication to mucosal surfaces. This is the same secretory protease which acts in NDV-LS in humans and non-human primates, limiting viral replication to the upper airways.

It is of note that the NDV genome is non-segmented. For this reason, transcription results in a single-stranded RNA which provides the genome with enough stability to avoid reassortment events. These features underpin the antigenic and genetic stability that have contributed to the success of NDV across decades as a vaccine vector.

The recombinant nature of the viral vector is based in the design and synthesis of a gene that codes for the spike protein in SARS-CoV-2. Such design is based in the sequence of the Wuhan-Hu-1 virus (NC_045512.2) and assembled in silico.

Lentogenic strains like LaSota have been used for more than 70 years of vaccination in avian populations and have proven to be safe and with a remarkable naturally attenuated viral activity. In fact, studies have shown that the insertion of foreign genes into the NDV genome leads to a further reduction of pathogenicity in birds. Furthermore, the rNDV is not excreted in feces and therefore not transmitted from bird to bird. Safety tests with avian rNDV vaccines have shown that doses 10 times higher than the dose suggested in this study are not associated with any pathogenicity.

A rNDV vaccine against SARS-CoV and other emerging infections had been proposed. It has been demonstrated that a rNDV vector expressing the S-protein in the SARS-CoV coronavirus is capable of developing protective immunity without safety concerns when administered to African green monkeys by the intranasal route.

It has been reported that rNDV injected by the IV route in non-human primates (Macaca fascicularis) was not associated with any severe disease or abnormalities in hematological or biochemical lab values.

Recently, in the context of the COVID-19 pandemic, a vaccine based on a S-protein expressing viral vector of the Newcastle Disease virus (NDV) has been studied using both a wild type and a pre-fusion membrane-anchored vector format. These studies were performed in mice and hamster models with two administrations of the vaccine. The tested vaccines induced high levels of neutralizing antibodies when administered by the intramuscular route. Notably, these vaccine prototypes protected mice against a mouse-adapted SARS-CoV-2 challenge: neither viral load nor viral antigen were detected in the lungs.

To produce the rNDV, a cell line is transfected by plasmids that express the whole viral genome that contains the gene in question. The clone of the whole NDV genome is transfected with helper plasmids that code for the viral proteins N, P and L under the control of the bacteriophage T7 RNA-polymerase promoter. The chimeric virus is obtained from the culture and propagated in chicken embryo SPF of 10 days of age, until the original vaccine virus is generated.

The vaccine has been formulated for intranasal and intramuscular administration.

In our study, ninety healthy volunteers with no history of COVID-19, vaccination against SARS-CoV-2 or an activity associated with a higher risk of exposure to SARS-CoV-2 will be assigned sequentially into one of nine treatment groups at a single research site in Mexico City.

These treatment groups correspond to three different doses and three different schemes of administration. All these schemes foresee two vaccine administrations separated by 21 days. 3rd administration by the intramuscular route to all the volunteers who agree to participate (see "Arms and Interventions").

Patients will be followed for efficacy and safety measurements. Efficacy will be measured by circulating and neutralizing IgG and IgM antibodies against the S protein of SARS-CoV-2, IgA titers in nasal mucosa and cytokine-mediated T cell responses. Patient safety will be monitored by the collection of information on adverse events and safety laboratory assessments (mainly hematology and blood chemistry).

The first intervention for each treatment group will be administered in a sequential way to eighteen sentinel subjects. Once all sentinel subjects have received the first intervention and the Safety Data Monitoring Board has determined that safety conditions have been met, the study will proceed to enroll the rest of the subjects until a total of 90 participants is reached.

Statistical tests will be applied to each treatment group with similar baseline characteristics. For continuous variables Student's t distribution and ANOVA will be used to compare mean values, while chi-square and Fisher´s exact test will be used to assess categorical values.

There are three working hypotheses to be tested, one for each scheme of administration. They can be consolidated as follows : The recombinant anti-SARS-CoV-2 vaccine based on a viral vector (rNDV) administered [two times by the intramuscular route / two times by the intranasal route / the first by the intranasal route and the second by the intramuscular route] is safe (i.e. an acceptable low profile or reactogenicity: low frequency of mild-to-moderate and no severe local or systemic adverse reactions) and induces a humoral and cellular immune response against SARS-CoV-2 similar (or greater) to that measured in sera from naturally-acquired COVID-19 convalescent individuals.

Inclusion Criteria:

• Adult men and women ≥18 year-old and ≤55-year-old.
• Signed informed consent.
• No respiratory disease within last 21 days prior to first dose administration.
• Body Mass Index from 18.0 to 29.0 kg/m2.
• Negative RT-PCR for SARS-Cov-2 infection.
• Negative test for anti-SARS-CoV-2 IgM and IgG antibodies.
• O2 saturation ≥92% by pulse oximetry.
• Normal CT scan of thorax.
• No symptoms from clinical history and normal physical exam at screening visit.
• Lab test values within normal ranges for all the following:

Urinalysis. Liver enzymes. Renal function tests. Cholesterol and Triglycerides. Fasting glucose. Hematology.

• Negative test for HBsAg, anti-HCV and anti-HIV antibodies. Negative VDRL test.
• Normal electrocardiogram.
• Negative pregnancy test for women with childbearing potential.
• Agreement of all sexually- active volunteers to use highly effective contraceptives over the study period and up to 30 days after the last administration of the experimental vaccine.
• Commitment from all participants to keep social distancing, use of mask and frequent hand washing with soap or antibacterial gel during the study period.

Exclusion Criteria:

• History of hypersensitivity or allergy to any ingredient of the vaccine.
• History of severe anaphylactic reaction.
• History of seizures.
• History of chronic diseases or cancer.
• Vaccination against SARS-CoV-2 with approved or experimental vaccines.
• Participation in any other study with an experimental intervention within the last 3 months.
• Administration of any other drug or herbal preparation within the last 30 days.
• Any vaccine administered within the last 30 days, including influenza vaccine.
• Fever at the time of entry.
• Blood transfusion or blood components transfusion within the last 4 months.
• Regular activity related to work, social interaction or entertainment that represents an exposure to SARS-Cov-2 higher than that of the general population, as per investigator judgement.
• Drug and alcohol abuse.
• Any medical or not medical condition that could interfere with patient safety, study compliance or data interpretation, as per investigator judgement.