7 Ad26 vectors were produced expressing I). tPA.S Tissue plasminogen activator (tPA) leader sequence with full S; II). tPA.S.PP (2 proline substitutions); III). S. Wild type leader with full S; IV). S.dCT Wild type leader with deleted C-terminal of S; V). tPA.WT.S tandem tPA and full S; VI). S.dTM.PP Wild type leader with deletions in transmembrane and cytoplasmic regions; VII). S.PP Wild type leader with with 2 proline substitutions and mutation at cleavage site. An empty Ad26 vector was used as a negative control. Transduction was successful and the presences of all recombinant antigens were detected from cell lysates in Western blot. Quantification of neutralizing antibody (NAb) responses was performed using live virus neutralization assay and pseudovirus neutralization assay. 
	Unlike other inactivated virus and nucleic acid vaccines, adenovirus vectors can induce long lasting Nab responses upon a single dose. However, it would be possible that an additional dose can enhance immunogenicity. Among the 7 S antigen vectors, the Ad26-S.PP was able to induce strong Nab responses and provide full protection against COVID-19 infection, but was weak to induce T-cell responses for adaptive immunity. Further studies regarding to the T-cell responses and half-lives of Nab were still yet to be determined. 


Ad26 Vector Transduction & Immunogenicity

Collaboration between Johnson and Johnson (J&J) and Janssen Pharmaceutical in addition with Beth Israel Deaconess Medical Center had recently tested a potential vaccine candidate detonated as Ad26.COV2.S. This vaccine utilized Janssen’s AdVac technology in engineering adenoviruses to infect hosts with COVID-19 antigen spike protein (S) to stimulate immunogenicity and neutralization through antibody secretion. Janssen’s previous AdVac program was successful in manufacturing Ad26.ZEBOV as a vaccine for the Ebola virus. Investigation of adenovirus serotype 26 (Ad26) was able to stimulate strong immune response in synthesizing neutralizing antibodies during preclinical trials in rhesus macaques. 52 of them were immunized with spike variants from Ad26 vectors and infected with COVID-19 through nasal and tracheal pathways, with Ad26.COV2.S as the best candidate.

Tufts University

So far, vaccines consist of either inactivated or weakened versions of antigens or virions. Many pharmaceutical companies like Pfizer and Moderna are developing inactivated antigens in the form of mRNA, DNA, and proteins from the viral envelope or capsid that are often fragmented. 

Vaccine Candidates, Immunogenicity, and Adaptive Immunity

Moderna was one of the earliest companies to propose potential vaccines after having screen through more than 120 mRNA-based molecules. Among them, they identified mRNA-1273 that is capable of triggering the adaptive immune response. This mRNA essentially codes for the viral spike (S) glycoprotein that is involved in receptor recognition with ACE2 receptors in host cells. In the pre-clinical trial experiments with Rhesus macaque, administration of either 10 or 100 micrograms of the mRNA-1273 showed successful induction of type 1 helper T-cells (Th1) and no detectable viral antigens over the course of the next few days. Immunohistochemistry results detected higher levels of IL-21 cytokines. These non-human primates over the course of 2 weeks also showed regression of upper and lower airway infection. 
In the study, there was elevated antibody-binding activity through ELISA detection as well as neutralization activity in vaccinated primates. mRNA-1273 lead to higher suppression of ACE2 binding recognition with spike protein. mRNA-1273 nucleoside modifications and purification were optimized to stimulate the innate immune system in order to enhance translation and duration. One remaining concern was the half-life of antibodies against spike protein. Recent human data shows a reduction of antibodies over time after initial infection. 


Moderna mRNA-1273

In the phase 1 and 2 clinical studies with BNT162b1, healthy participants from 18 to 55 years old will receive either the lipid nanoparticle-delivered vaccine at (10µg, 30µg, and 100µg) or placebo. The nucleoside-modified mRNA sequence codes for the spike glycoprotein that is trimerized. IgG antibody binding and spike protein neutralization was dosage dependent. The mRNA vaccine was relatively safe as it would not integrate into host genome and would be translated to produce spike antigen to trigger immune response. One particular enhancement was to add 1-methyl pseudouridine to increase translation efficiency while triggering innate immune response. 
The receptor binding domain (RBD) of BNT162b1 was further modified by the addition of a phage T4 fibritin-derived trimerization domain to increase its immunogenicity before being encapsulated in lipid nanoparticles administered through intramuscular injection. Around 7 days into vaccination, both the BNT162b1 and placebo recipients experienced mild to moderate levels of fatigue and headache, with more frequencies from the BNT162b1 participants. For the most part, no fatal symptoms seem to occur. Persistent follow up will need to be continued for up to half a year while immunogenicity will need to be taken for up to 2 years. Adults over 65 years old are also beginning to enroll in this clinical trial. 


Pfizer BNT162b1

The application of synthetic DNA medicine (INO-4800) in blocking the infection pathway of SARS-CoV-2 have already passed phase 1 of clinical trial on a positive note at Inovio as they now look forward to add participants of older age. Unlike RNA-based vaccine candidates, synthetic DNAs are much easier to design and amplify. They are also more temperature-stable and resistant to nucleases. Previous works done on the MERS-CoV vaccine with INO-4700 as well as Zika virus with GLS-5700 were quite successful in halting infections. These experiences show that INOVIO could potentially apply similar strategies to drive neutralizing antibody responses for long-term protection from COVID-19.
	In general, induction of INO-4800 to African green monkey fibroblast (COS-7) and human kidney (HEK-293T) cell lines both showed substantial levels of antibodies in serum. It is capable of activating cellular and humeral responses within several days post immunization. The receptor binding domain sequence of the spike protein was cloned into pGX9501 and pGX9503 vectors that are transfected into COS-7 and HEK-293T cells showed high levels of RNA levels upon detection by RT-qPCR. Spike protein expression was measured by Western blot to be consistently high and further confirmed qualitatively through immunofluorescence staining. INO-4800 seemed to exhibit competitive inhibition against ACE2 receptor and was also detected in the lungs that halted Lower Respiratory Disease (LRD).


Inovio INO-4800

J&J and Janssen Pharmaceuticals: AD26.COV2.S

In a study done by Oxford and Astrazeneca on an adenovirus vector vaccine candidate from chimpanzee (ChAdOx1 nCoV-19) expressing the spike protein, phase 1 and 2 were done on healthy adults from 18 to 55 years old with no prior exposure to COVID-19. ChAdOx1 (AZD1222) consisted of the entire spike sequence linked to a tissue plasminogen activator leader sequence. For placebo, some adults were administered a meningococcal conjugated vaccine (MenACWY) while the other were administered ChAdOx1 all with single intramuscular dosages. MenACWY was used as placebo in order to cover up the effects of receiving AZD1222. Previous clinical studies in Rhesus macaques show promising efficacy in inducing humoral/cellular immune responses as well as resistance toward lower respiratory tract infections.

Oxford-Astrazeneca: ChAdOx1

- Initiative
- Candidate requirements
- Strategy

Operation Warp Speed

Corum, J., Wee, S., & Zimmer, C. (2020). Coronavirus Vaccine Tracker. https://www.nytimes.com/interactive/2020/science/coronavirus-vaccine-tracker.html

Coronavirus Vaccine Tracker

- Novavex, in collaboration with Takeda, recently developed a protein-based vaccine candidate (NVX-CoV2373) that were constructed from the COVID-19 full-length spike protein, transfected into Spodoptera frugiperda (SF9, fall armyworm) cell lines, and purified. 
- A stable inactivated form of premature S-protein was constructed through mutagenesis by mutating the furin cleavage site as well as incorporating 2 proline substitutions that destabilize the central alpha-helix. 
- Administration of NVX-CoV2373 with Matrix-M adjuvant, a synthetic biomolecule consisting of saponins conjugated to cholesterol and phospholipids, compacted into nanoparticles have shown to induce strong immune responses like Th1 B-cell and T-cell responses as well as secretion of neutralizing antibodies in serum.
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- In preclinical trials, mice were administered in increasing doses of 0.01μg, 0.1μg, 1μg, and 10μg each with 5μg of the Matrix-M. Mice at all dosages exhibit high anti-spike IgG within 3-4 weeks post-initial vaccination. 
- Those vaccinated at 10μg even had complete inhibition of ACE2 receptor binding to spike protein. 
-Even with the smallest dosage, significant amount of inflammatory cytokines like interferon (IFN-γ), tumor necrosis factor (TNF-α), and interleukins (IL-2, IL-4, & IL-5) were secreted from effector memory T-cells. 
- Histopathology of lung tissues showed significant regression of lung tissue inflammation for those administered with NVX-CoV2372 compared to placebo. 
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Description from Lit Review Draft:

"NVX-CoV2373 is a recombinant nanoparticle vaccine composed of trimetric full-length SARS-CoV2 spike glycoproteins and Matrix M1 adjuvant. A randomized, placebo-control, phase 1-2 trial was conducted on 131 healthy adults.  It has high immunogenicity and high levels of neutralizing antibodies. On September 24, 2020, phase 3 clinical trials began and immunized 10,000 people in the next 4-6 weeks. This took place at the Nucleus Network in Herston, Queensland and Melbourne, Victoria.
This vaccine candidate is a recombinant nanoparticle vaccine constructed from full-length wild-type SARS-CoV-2 spike glycoprotein. It includes Matrix M1 Which is a saponin based adjuvant made by Novavax. The rSARS-CoV-2 and Matrix M1 parts were mixed just before use. This vaccine is favorable because it can be stored between 2°C and 8°C, allowing for easy preservation and, therefore, distribution (Novavax Initiates Phase 3 Efficacy Trial of COVID-19 Vaccine in the United Kingdom | Novavax Inc. - IR Site, n.d.)."

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