key: cord-0729397-hra6fxl7
authors: Batty, Cole J.; Heise, Mark T.; Bachelder, Eric M.; Ainslie, Kristy M.
title: Vaccine formulations in clinical development for the prevention of severe acute respiratory syndrome coronavirus 2 infection
date: 2020-12-13
journal: Adv Drug Deliv Rev
DOI: 10.1016/j.addr.2020.12.006
sha: a527b4d23864c33f9b56c5cd685891fd8c23a6f3
doc_id: 729397
cord_uid: hra6fxl7

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to an unprecedented effort toward the development of an effective and safe vaccine. Aided by extensive research efforts into characterizing and developing countermeasures towards prior coronavirus epidemics, as well as recent developments of diverse vaccine platform technologies, hundreds of vaccine candidates using dozens of delivery vehicles and routes have been proposed and evaluated preclinically. A high demand coupled with massive effort from researchers has led to the advancement of at least 31 candidate vaccines in clinical trials, many using platforms that have never before been approved for use in humans. This review will address the approach and requirements for a successful vaccine against SARS-CoV-2, the background of the myriad of vaccine platforms currently in clinical trials for COVID-19 prevention, and a summary of the present results of those trials. It concludes with a perspective on formulation problems which remain to be addressed in COVID-19 vaccine development and antigens or adjuvants which may be worth further investigation.

replication and had the highest virus yield when cultured in vitro in Vero cells [47] . Vero cells have been certified by the WHO for use in vaccine production and have previously been used to generate polio and rabies virus for vaccines. In comparison to virus from other patients, the HBO2 strain had 100% homology in the Sprotein. HBO2 was passaged ten times in Vero cells to induce adaptation to the host Vero cells. The tenth passage was deep sequenced and showed a 99.95% homology to the 7 th passage, with a 100% homology to the amino acid sequence of the S-protein of the 7 th passage, indicating that the virus had adapted and reached a stable genetic sequence, rendering it suitable for further scale up. This strain was mass produced in Vero cells using a novel basket reactor and inactivated by the addition of β-propionolactone. The resulting inactivated virus was then mixed with aluminum hydroxide (alum) adjuvant in bulk prior to administration.

To evaluate the alum-adjuvanted inactivated virus as a vaccine candidate, Wang et al. immunized mice, rabbits, rats, guinea pigs, cynomolgus monkeys, and rhesus macaques, resulting in 100% of the animals having detectable antibodies (seroconversion) 21 days after immunization. Additionally, rhesus macaques immunized with the alum-adjuvanted inactivated virus showed no viral load in the lungs. In other organs, viral load in the vaccinated group was much lower compared to the unvaccinated controls. Additionally, there was no detectable ADE after infection.

The safety and immunogenicity of this formulation in humans were subsequently evaluated in Phase 1 and 2 clinical trials [48] . Interestingly, neutralizing antibody titer as measured by the Plaque Reduction Neutralization Tests (PRNT) assay, as well as SARS-CoV-2-specific IgG titers, did not show a dosedependence between the low, medium, and high dosages administered during Phase 1. Adverse events also did not show a dose dependence, and included fatigue, fever, nausea, and pain and swelling at the injection site. Higher neutralizing and antigen-specific IgG titers were elicited from groups receiving a boost vaccination 21 or 28 days after the prime injection as compared to those boosted 14 days after prime injection. While this study did not include comparisons to convalescent serum, the results indicated that a significant neutralizing and SARS-CoV-2 specific antibody response could be raised in humans with this formulation. Phase 3 clinical evaluation of this candidate is now underway (ChiCTR2000034780).

Another Chinese company, Sinovac, has published on their inactivated SARS-CoV-2 virus with similar results, eliciting 92.4% seroconversion using a day 0 and 14 prime-boost schedule and 97.4% seroconversion by a Day 0 and 28 prime-boost schedule [49, 50] . Despite the evidence for greater seroconversion with a boost J o u r n a l P r e -p r o o f Journal Pre-proof at day 28 rather than day 14, they have since begun evaluating a day 14 boost schedule in Phase 3 clinical trials in Indonesia (INA-WXFM0YX), Turkey (NCT045823440), and Brazil [51] (NCT04456595).

Overview of nanoparticulate subunit technologies applied to SARS-CoV-2 vaccines. [52] [53] [54] [55] [56] [57] [58] [59] [60] [61] [62] 

Subunit formulations are a common vaccine type where one or more elements of the pathogen are used as an antigen. These elements can be proteins, peptides, or sugars of the pathogen. Additionally, nucleic acids such as mRNA or DNA can be administered to use the body's protein expression machinery to express subunit elements of the pathogen. Because subunit vaccines are only elements of the pathogen, they are considered the safest type of vaccine. One issue, however, is that they are often poorly immunogenic and Table 1 

Another form of multimerized recombinant protein antigen is the RBD-dimer [83] . This antigen was initially developed upon observation that a recombinant RBD of MERS existed in an equilibrium state between monomers and dimers in solution, and that the dimer exhibited significantly greater immunogenicity compared to the monomer upon immunization in BALB/c mice. To create a stable form of this dimer, cysteines which usually formed a disulfide bridge at the C' terminus of each of the RBD constructs were truncated and then connected in tandem as a single-chain construct. An analogous strategy was subsequently used to generate RBD-dimers of SARS-CoV and SARS-CoV-2. The resulting single chain constructs exhibited higher RBDspecific IgG and pseudovirus neutralization titers relative to their monomers after immunization of BALB/c mice in combination with AddaVax (MF59-like) adjuvant. However, in the case of the SARS-CoV-2 RBD-dimer, splenocytes isolated 45 days after the last of three vaccinations did not demonstrate significant secretion of IFN-γ, IL-2, TNF-α, or IL-4 after stimulation with an RBD-derived peptide pool, indicating a poor induction of Vaxine Pty Ltd. have partnered with Medytox in the development of an adjuvanted subunit protein vaccine. To our knowledge, the only publicly available information regarding the antigen being employed is that it is a recombinant spike protein [40] , however, the adjuvant being employed is Advax-SM. Advax is composed of the immunostimulatory polysaccharide delta-inulin, which has been demonstrated to amplify the immune response without affecting Th1/Th2 skew when co-administered with diverse antigens such as those from influenza and hepatitis B [84] . In addition, intramuscular prime-boost immunization with SARS-CoV spike protein and Advax or Advax and CpG induced spike-protein specific neutralizing antibody titers in BALB/c mice [85] . Using SARS-CoV spike protein to stimulate splenocytes harvested 1 year after vaccination, mice immunized with Advax alone demonstrated significantly greater secretion of IFN-γ, IL-2, and IL-4 compared to those immunized with spike protein alone, or those immunized with spike protein, Advax, and CpG. In the same experiment, the Advax and CpG adjuvanted group demonstrated significantly greater secretion of IL-17 compared to all other groups. In addition, both Advax-containing groups were protected from lethal challenge with mouse-adapted SARS-CoV and exhibited reduced eosinophilic immunopathology in the lung compared to mice immunized with spike protein and alum. This raises the possibility that a Th1 and/or Th17-skewed T cell responses may help to reduce immunopathology upon SARS-CoV infection, which merits further investigation with SARS-CoV-2. Despite the prime-boost schedule employed in this preclinical study, the Phase 1 clinical trial will use a prime-only intramuscular vaccination (NCT04453852).

Medigen Vaccine Biologics Corporation, the National Institute of Allergy and Infectious Disease (NIAID), and Dynavax are collaborating to advance a vaccine candidate employing a spike protein with a double proline (2P) substitution known to stabilize coronavirus spike proteins in their prefusion conformations [86, 87] . This antigen was evaluated preclinically in mice in combination with alum and CpG 1018, and shown to generate anti-spike and pseudovirus-neutralizing titers from a prime-boost regimen [88] . The T cell response J o u r n a l P r e -p r o o f Journal Pre-proof to this antigen with alum, CpG, or the combination also indicated that the addition of CpG to alum reduced the Th2 skew compared to alum alone.

While soluble antigens represent a relatively simple form of antigen which is employed in many currently-approved vaccines, arranging protein antigens into a particulate form offers the potential advantages of greater B cell activation by increased B cell receptor crosslinking, increased cross-presentation of particulate antigens, and a greater likelihood of receiving T cell help with the codelivery of multiple proteins with potential T cell epitopes. [81] .

The stability of the engineered antigen in stressed storage conditions was also assessed. This is a highly important facet to investigate, as stability of a vaccine formulation can significantly diminish logistical hurdles, especially if the formulation can demonstrate stability outside of cold chain conditions [95] . Stability was assessed by exposing the antigens to either prolonged agitation, elevated temperature (25 or 37 °C), pH (4 or 9), or oxidating conditions by hydrogen peroxide, each for 48 hours. Of these conditions, only oxidizing conditions affected the binding affinity of the stabilized spike trimer to hAce2 in an ELISA experiment, while the trimer lacking the stabilizing 2P modification had reduced binding affinity from multiple stress conditions. This indicated the stabilizing effect of the 2P modification and gives an initial indication of stability of this formulation, although much more rigorous stability studies must be undertaken to understand the stability of any vaccine formulation approved for human use [96] .

Vaccination of mice with this stabilized spike trimer nanoparticle antigen along with Matrix-M adjuvant generated a high spike-specific titer, significant CD4+ and CD8+ antigen-specific response and a Th1 dominant phenotype and protected against mouse-adapted viral challenge. Similarly, vaccination of olive baboons showed generation of high titers of anti-spike IgG, in groups which received both the protein nanoparticle antigen and the adjuvant. A subsequent study demonstrated that prime-boost vaccination with this formulation inhibited SARS-CoV-2 replication and pathology in the upper and lower airways after administration of the virus by intranasal and intratracheal instillation [97] .

The Novavax vaccine formulation was then evaluated in a Phase 1/2 clinical trial (NCT04368988).

Using a prime-boost schedule with the boost occurring at day 21 after the prime, they detected significant formation of spike-specific and viral neutralizing antibodies [98] . This result was not significantly dosedependent, but inclusion of the Matrix-M adjuvant significantly enhanced overall anti-spike IgG titer and viral J o u r n a l P r e -p r o o f Journal Pre-proof neutralizing titer. In the subset of patients who were evaluated for a T cell response, significant Th1-skewed spike-specific responses in CD4+ T cells were noted 7 days after the boost in groups receiving the adjuvanted vaccine. Also, of note was the fact that this formulation was stored at 2-8 °C, an easier condition to maintain than subfreezing conditions stipulated by other candidate vaccines.

Novavax is conducting a second Phase 2b clinical trial in South Africa in collaboration with the Bill and Melinda Gates foundation (NCT045333990). Notably, this trial will recruit approximately 240 HIV-positive patients to evaluate the safety and immunogenicity of the vaccine in this highly vulnerable immunocompromised population, in addition to gathering further safety, immunogenicity, and preliminary efficacy data in healthy HIV-negative participants. Novavax has begun a Phase 3 clinical trial in the United Kingdom (2020-004123-16) and is slated to begin further Phase 3 trials in the United States and Mexico.

A VLP is a viral particle that displays protein antigens, that lacks any DNA or RNA, and maintains the structure of the original virus particle [99] . Based on their surface, size, and shape, VLPs can display epitopes in a highly dense fashion which allows for potent stimulation of the immune system [100] . Most VLPs are made recombinantly in mammalian cells. However, the company Medicago uses plants as a source to produce recombinant proteins that self-assemble into VLPs. Growing proteins in plants for vaccine applications is inexpensive, and inherently has a low risk of contamination with mammalian pathogens, or endotoxin [99] .

Researchers use the bacterial vector, Agrobacterium tumefaciens, to transiently infect plants by forcing a bacterial suspension into the extracellular space of the leaf tissue [101] . Nicotiana benthamiana, a close relative of the tobacco plant indigenous to Australia [102] , is used extensively to produce recombinant proteins since it allows a wide range of pathogens to infect it. Medicago has previously published a Phase 2 clinical trial using their system for a quadrivalent plant-derived VLP influenza vaccine [103] . They showed that the incidence of pain at the injection site is higher than compared to placebo. However, most of the local symptoms were mild and resolved within a day. The addition of alum with VLPs did not increase the antibody titers. In measuring the antibody response, they did not use a positive control based on an inactivated influenza vaccine, so it is hard to determine how effective the VLP technology is compared to traditional methods. Medicago was able to generate cross-reactive antibodies against heterologous strains of influenza and illustrate T-cell responses, which potentially would allow for broad protection. Based on this technology, J o u r n a l P r e -p r o o f

Medicago has announced that they are in the process of going into Phase 1 Clinical Trials for a COVID-19

vaccine (NCT04450004) [62] .

Nucleic acid based vaccines were first identified in the early 1990's when plasmid was injected intramuscularly and a humoral response to the encoded antigen was noted [104] . Quickly thereafter, clinical trials began for cancer and infectious disease vaccines, wherein plasmid was introduced via electroporation or injector gun. With DNA based vaccines, the plasmid must be delivered to the nucleus of the host cells to induce antigen expression and the resulting immune response. Shortly after DNA vaccines were identified, mRNA-based vaccines were developed [105] . In contrast to DNA based vaccines that require nuclear delivery, mRNA vaccines only need to be delivered to the cytoplasm. Moreover, although it has never been reported, DNA vaccines do theoretically have the potential to incorporate into the host genome, a problem that is avoided with mRNA vaccines. On a molar basis, DNA and mRNA vaccines are theoretically similarly efficacious; however, mRNA can lead to more rapid expression than DNA [105] . Overall, the first generation of DNA and RNA based vaccines did not illustrate strong protection in humans. Advancements in the use nucleic acid vaccines have focused on better identification of antigens, modifications to improve nucleic acid stability and translation, improved delivery, and inclusion of adjuvants to generate more protective responses.

With respect to delivery of RNA and DNA based vaccines, neutralization of the negative charge of the nucleotides can facilitate delivery through the cell membrane. To this end, cationic polymers, proteins, lipids, or other elements have been used to form complexes with anionic nucleotides to generate non-viral vectors for RNA or DNA delivery. For the portion of negative to positive charges, often an N/P ratio is reported to relate the number of amine groups on the cationic material that can be positively charged to the number of nucleotide phosphate groups on the that can be negatively charged. Changing the N/P ratio of the carrier and the nucleic acid can influence many other properties such as the stability, size, and net surface charge. Usually increasing the N/P ratio increases activity. However, there is a trade-off with an increase in activity, which is an increase in toxicity. An increase in the N/P ratio helps particles enter the cell, open the phagosome/lysosome, and allows the nucleic acids escape to enter the cytosol for RNA, or the nucleus for DNA [106] . One mechanism by which this occurs is the 'proton sponge' effect, where a weakly basic molecule causes the phagosome to leak and perhaps rupture [107] .

J o u r n a l P r e -p r o o f

Of all the technologies applied for SARS-CoV-2, nucleic acid vaccines have seen the most rapid advancement to clinical evaluation. This is indicative of one significant advantage to this technology, the ease and speed of development. If a company already has a gene delivery platform, then as soon as an antigen's genetic sequence is known, a vaccine can be developed. One drawback with this technology is that although there has been research on DNA or RNA vaccines for upwards of 30 years, there has yet to be an FDA approved formulation that is available clinically.

Electroporation is a method that Inovio Pharmaceuticals is pursuing for COVID-19 vaccines [53] . In general, electroporation is the application of brief electric pulses to cells and tissue which transiently and reversibly permeabilize the cell membrane. This disruption allows for the entry of large molecules, including plasmid DNA to enter the cell. Using electroporation can drastically enhance the protein expression generated by the injection of a plasmid [108] . Enhancement of the immune response using electroporation could be in part due to the induction of a local inflammatory process. Electroporation induces the production of inflammatory cytokines and chemokines along with the recruitment of immune cells [109] .

Previously Inovio Pharmaceuticals used electroporation to deliver a DNA vaccine against MERS in a Phase 1 clinical trial [110] . Vaccination involved using the plasmid, GLS-5300, a DNA vaccine expressing a full-length MERS coronavirus S-protein. Vaccination involved injecting the plasmid in one mL intramuscularly in the deltoid followed by intramuscular electroporation at the site of injection to enhance plasmid entry, using a Cellectra-5P Adaptive Constant Current Electroporation Device, which is made by Inovio Pharmaceuticals.

This device emits square-wave electric pulses with an adjustable electric field [111] . In their Phase 1 clinical trial against MERS, 50% of the vaccinated participants generated detectable neutralizing antibodies at one or more timepoints in the study. Additionally, 88% of the patients had T cells that produced IFN-γ in the presence of the S-protein.

With regards to COVID-19, Inovio Pharmaceuticals generated preclinical data showing the efficacy of their vaccine [112] . A plasmid was generated encoding the SARS-CoV-2 S-protein along with a N-terminal IgE leader sequence. The plasma generated contained a human cytomegalovirus (CMV) immediate-early promoter and a bovine growth hormone polyadenylation signal. Using their system to vaccinate BALB/c mice and guinea pigs resulted in the generation of neutralizing antibodies against SARS-CoV-2, showing the immunogenicity of J o u r n a l P r e -p r o o f Journal Pre-proof their system. They also reported generation of neutralizing antibodies and IFN-γ-secreting spike-specific T cells in rhesus macaques after a prime-boost immunization, leading to a significant reduction in viral load, relative to naïve controls, in bronchoalveolar lavage fluid and nasal swab samples collected after intratracheal and intranasal challenge with SARS-CoV-2 17 weeks after vaccination [113] . On April 6, 2020, Inovio

Pharmaceuticals announced they will start Phase 1 clinical trials [52, 53] .

Another company, Genexine, has also reported the start of a Phase 1/2 clinical trial against COVID-19

[114]. Genexine has previously published results from Phase 1 and Phase 2 clinical trials using electroporation for HPV vaccination [115, 116] . The plasmid encoded the virus antigens HPV E6 and E7. Additionally, unique to the other nucleic acid approaches listed in this review, the plasmid encoded an adjuvant: Fms-like tyrosine kinase-3 ligand (FLT3L). A previous study showed that a nucleic acid cancer vaccine can be boosted with the addition of FLT3L [117] . Systemic delivery of FLT3L ligand prior to injecting RNA increased T-cell homing to the tumor and the vaccine's therapeutic efficacy. Overall, the cure rate was enhanced by the addition of FLT3L.

In the Phase 2 clinical trial that Genexine ran using their electroporation system, 63% of the patients showed histopathological regression. Unfortunately, no placebo was used, and no historical controls were discussed in the publication. In a recently released preprint, Genexine detailed that they compared intramuscular vaccination of BALB/c mice with DNA coding for the full-length S protein (pGX27-S) or the S protein without the S2 portion (pGX27-S ΔTM ), and found that pGX27-S ΔTM induced higher anti-S protein titers, leading them to choose this antigen for further evaluation in vaccination of rhesus macaques. Using a prime-boost-boost model in rhesus macaques, they saw induction of anti-S and neutralizing IgG in all animals after one vaccination, with titers increasing after the boosts. They also saw induction of S-specific CD4 and CD8 t cells. They saw an insignificant reduction in viral loads relative to controls after viral challenge ten weeks after the last vaccination, but saw a significant reduction in airway tissue pathology 4 days after infection relative to unvaccinated controls [118] .

An alternative strategy for DNA transfection of the skin is being employed in a collaboration composed of Osaka University, AnGes, and Takara Bio. While publicly available information on the antigen and adjuvant is unavailable, it is evident that they are employing plasmid DNA delivered by a pyro-drive jet injector (PJI), which employs the detonation of small amounts of explosive powder to propel plasmids in a jet into the skin at variable, controllable depths [119] . In a preclinical evaluation of this technology to vaccinate against the model J o u r n a l P r e -p r o o f Journal Pre-proof antigen ovalbumin in mice and rats, they demonstrated that plasmid DNA delivered by PJI induced significantly greater antigen expression and resultant antibody titers compared to needle-injected plasmid DNA [120] . This technology presents the advantage of needle-free, single step delivery with the potential to optimize the depth of delivery to induce the optimal immune response. Clinical Phase 1/2 evaluation of the PJI system delivering a COVID-19 vaccine is currently underway (NCT04463472).

TranslateBio and Sanofi have entered an agreement to develop a vaccine that will enter Phase 1 clinical trials in the fourth quarter of 2020 [121] . compared to a linear polymer, allowing for the facile delivery using a vibrating mesh nebulizer. They showed that covalent attachment of polyethylene glycol (PEG) to the polyplex did not increase stability of the particle, and therefore they did not move forward with in vivo delivery using a PEGylated formulation. Using their system, Patel et al. were able to obtain expression in all lobes of the lung in mice, and a majority of cells transfected were lung epithelial cells. Protein expression plateaued at 24 hours but quickly dissipated at 48

hours. Using this system, TranslateBio and Sanofi will hopefully develop a vaccine that will enter Phase 1 clinical trials soon for SARS-CoV-2.

Due to its hydrolytic instability, poor membrane permeability, and the abundance of RNAses in the body, mRNA must generally be complexed for otherwise protected to ensure delivery to the cytosol where it can result in translate of its encoded antigen. One method to accomplish this is through LNPs. Nucleic acid containing LNPs can be formed by mixing mRNA, various ionizable and neutral lipids. The lipids and nucleic J o u r n a l P r e -p r o o f Journal Pre-proof acids are often mixed with a microfluidic mixer or other similar mixing apparatus [54] . Ionizable lipids have a pK a such that they are positively charged at a low pH, allowing for complexation of anionic RNA, but neutral at neutral pH, reducing their toxicity relative to cationic lipids. Moderna is a company which has been working to use this delivery method for the treatment of a broad spectrum of diseases. [125] . Titers increased with the boost and the upper 50% of the neutralizing titer were comparable to a panel of convalescent serum samples. Adverse events increased with the boost, with 20% of the participants in the 250 µg dose reporting one or more adverse events. Subsequently, this trial was expanded to include 40 participants over the age of 55 [126] . These patients were only administered the lower (25 μg and 100 μg) dosages due to their lower incidence of adverse events in the younger cohort. No serious adverse events occurred in this trial [127] , and the most common adverse events were headache, fatigue, myalgia, chills, and injection-site pain. Most of these adverse events were elicited after the boost of the [128] . Moderna's trial Phase 3 Trial has been noted for its diversity with 37% of the study representing communities of color (e.g. African American, Hispanic). In addition, to greater than 42% of individuals in the study are from at least one high-risk group (e.g. obese, diabetic, 65+) [129] .

As stated previously, an advantage of the LNP system is the speed at which it can be developed. The day after SARS-CoV-2 sequence was released publicly, the modified prefusion sequence was determined, and synthesis was started. [131] and indeed Moderna has already filed for emergency FDA approval of their vaccine. [132] J o u r n a l P r e -p r o o f

Another LNP vaccine is being developed via a partnership between Pfizer and BioNTech [133] .

BioNTech has previously published a preclinical study in animal models of Zika virus [134] . and BioNTech initially reported a greater than 90% interim vaccine efficacy with their formulation [139] , which J o u r n a l P r e -p r o o f Journal Pre-proof was later revised to be 95% effective in preventing symptomatic COVID-19 [140] . Recently, Pfizer/BioNTech has been approved for application in the United Kingdom [141] .

Another mRNA lipid nanoparticle under development as a COVID-10 vaccine is formulated by CureVac. Classically, CureVac has reported the complexation of mRNA with protamine. Using this protamine approach Curevac was the first to show the use of an mRNA vaccine nanoparticle in humans [58] . Curevac has announced that they will begin Phase 1/2a clinical trials in June of 2020 for a SARS-CoV-2 vaccine, with this vaccine utilizing the same lipid formulation as Pfizer/BioNtech with mRNA encoding S protein of the virus [58, 143, 144] . This change from a protamine based carrier to a lipid based particle may be due to the fact that LNPs are shown to work better when given via standard intramuscular injection [145] , whereas the protamine based formulation only induced neutralizing titers when administered with less common needle-free injection mechanisms similar to those used in previous studies of DNA vaccination [58, 120] . Another consideration might be the enhanced storage they announced with their new LNP formulation, noting that they have a formulation suitable for storage at three months at standard refrigeration temperature (5 ºC) [146] .

Another RBD-encoding mRNA LNP formulation was developed by Zhang et al. [147] . Additionally, there are other LNP systems for delivering mRNA. An Imperial College London laboratory has avoided collaborating with any major pharmaceutical company in order to deliver a vaccine to the United Kingdom and developing countries for a reasonable price [149] . This group is using a self-amplifying RNA approach. Self-amplifying (or replicating) mRNA (also called a replicon) encodes the antigen of interest as well as proteins which lead to replication of the subgenomic RNA encoding for the antigen. Rather than just the sequence encoding the antigen, the mRNA sequence is a single strand that is able to self-replicate in the cytoplasm of the cell [150] . Accordingly, less self-amplifying mRNA is theoretically needed compared to traditional mRNA. Imperial College London's RNA vaccine is formulated into lipid nanoparticles with cationizable lipids similarly to Moderna and Pfizer, using a cationizable lipid patented by Acuitas Therapeutics [151] . They have recently published their preclinical results, wherein a prime-boost immunization induced high levels of Th1-biased virus-specific and neutralizing titers and SARS-CoV-2 peptide-specific T cells in mice [152] . The Imperial College London group is currently conducting a Phase 1 clinical trial with this formulation using a prime-boost schedule (ISRCTN17072692).

Duke-National University of Singapore and Arcturus Therapeutics are also developing a replicon RNAbased vaccine [153] . They are delivering a replicon encoding the spike protein using their proprietary Lipidenabled and Unlocked Nucleomonomer Agent modified RNA (LUNAR) formulation. This formulation contains common components of LNPs for mRNA delivery such as cholesterol, phospholipids, PEGylated lipids and a

proprietary lipid with an ionizable amino head group [154] [155] [156] . In addition, this lipid has an ester group incorporated into it to facilitate rapid degradation after RNA delivery. The U-N-A portion of the acronym refers to unlocked nucleomonomer agents, which are nucleotides lacking a carbon-carbon bond between their 2' and J o u r n a l P r e -p r o o f Journal Pre-proof 3' carbons, and potentially including rearrangement of the location of the phosphoester bond relative to those carbons, or substitution with new functional groups at those carbons. These modifications are undertaken to alter the physicochemical, and potentially translational, properties of RNA molecules containing UNAs [157] .

However, there is no published data available on the extent to which these UNAs are incorporated into the Arcturus vaccine candidate RNA sequence. While preclinical data has not yet been reported for this formulation, Phase 1/2 clinical evaluation has begun (NCT04480957). The combination of the two strategies resulted in a virus which induced robust immunity in mice, including aged mice, to heterologous CoV challenge while also resulting in no pathology. Some groups have also preliminarily reported the isolation of attenuated SARS-CoV-2 mutants [166] , and intranasal inoculation of Syrian golden hamsters with this virus resulted in significant viral replication in the nasal turbinates but not in the lungs, milder lung tissue pathology than a SARS-CoV-2 control, development of SARS-CoV-2 neutralizing serum antibodies, and sterilizing immunity against SARs-CoV-2 infection [167] . At least three other groups are working on the generation of attenuated SARS-CoV-2 vaccines through the use of codon deoptimization to create viruses attenuated through reduced mRNA stability and translation efficiency [40, 168] .

As viruses and viral vectors have advanced, particularly for application in gene therapy, platforms have been developed that can be used to plug-and-play genetic information for treatment of a specific disease or development of a vaccine. In much the same way that non-viral vector vaccines can be accelerated into CanSino has previously shown the efficacy of using a replication defective adenovirus type 5 (Ad5)

vector as an Ebola vaccine in a Phase 2 clinical trial [171] . [172] . Another concern about using adenovirus is preexisting immunity against the virus in the general population. Of the patients enrolled in this clinical trial, 85% had preexisting antibodies against Ad5. Even though these antibodies existed, vaccination with the platform generated antibodies against Ebola on day 14, 28, and 168 when the study was conducted in China.

With these results, CanSino is developing a vaccine against SARS-CoV-2, and they were the first company to publish the results of their Phase 1 clinical trial [173] . For this trial, they developed an Ad5 virus expressing the S-protein from SARS-CoV-2. Patients were administered a single (prime-only) intramuscular injection of the viral vector. Similar to the Ebola vaccine, patients in the trial had a preexisting immune response against Ad5. However, for patients who received the high dose of virus particles, 94% still produced an immune response on day 14, while 100% produced an immune response on day 28. Furthermore, 75% of the high dose group had a four-fold increase in neutralizing antibody by day 28 compared to patients who did not receive the vaccine. It was noted that high preexisting antibodies did lower the immune response against the S-protein. These patients will be continued to be monitored to measure long term memory responses generated by this platform.

The results of a Phase 2 trial have also been reported for the CanSino Ad5 vaccine candidate [174] .

Patients were again administered a prime-only immunization consisting of one of two different doses (5 x 10 10 and 1 x 10 11 viral particles) injected intramuscularly. RBD-specific and virus-specific neutralizing antibodies were elicited at similar levels in both groups by day 28 after injection. However, individuals with pre-existing anti-Ad5 antibodies (52% of all participants), and those older than age 55 both demonstrated significantly lower titers. Spike-specific T cell responses were determined using an ELISPOT for IFN-γ secreting T cells.

Journal Pre-proof Significant increases in these cells were detected by day 28 in ~90% of participants, with no significant differences between those with and without preexisting anti-Ad5 antibodies, or between age groups. In addition, no serious adverse events were observed, with common adverse events such as pain, fatigue, and fever being observed in both dose groups. These results have led CanSino to pursue evaluation of this candidate in a Phase 3 trials (NCT04526990, NCT04540419), again using a prime-only immunization schedule. It remains to be seen whether the reduced humoral response in patients with preexisting anti-Ad5 immunity will hamper the protective efficacy of this candidate.

Other companies are also using adenovirus for vaccination against COVID-19. Oxford is collaborating with AstraZeneca in developing an COVID-19 using an adenoviral vector derived from a chimpanzee adenovirus and encoding the S protein (ChAdOx1 nCoV-19) [175] . Because the vector is derived from chimpanzees rather than a human adenovirus, pre-existing ant-vector immunity is very low in the general population. Preliminary results of the Phase 1/2 clinical trials that started April 29, 2020 have been reported, wherein ChAdOx1 nCoV-19 was compared with a meningococcal conjugate vaccine as control [176] . The vaccine was administered on a prime and boost (day 0, 28) schedule with prophylactic acetaminophen given, which reduced local and systemic reaction to the vaccine. After a single vaccination, 91% (32/35) patients had neutralizing antibody titers, and with boost, 100% of participants had neutralizing responses. Neutralizing titers correlated well with total anti-spike IgG antibody levels. They report T-cell response to the spike protein, noting it peaked at day 14, prior to the boost [176] . In November of 2020, AstraZeneca reported a 70% efficacy with their vaccine. Interestingly, two different doses were given at their two trial sites with increased efficacy noted at the site which administered a lower initial dose. The half-dose prime followed by a full-dose boost resulted in a 90% efficacy, whereas patients which received a prime-boost with a full dose reported a 62% efficacy. This disparity in efficacy could be due to an adverse immune response to the viral vector, rendering the boost less effective due to neutralization generated from the prime vaccination [177].

Janssen's AdVac® technology uses a nonreplicating version of adenovirus type 26 (Ad26) and has been used as a platform against the Ebola virus [178] . Neutralizing antibody titers against Ad26 are much lower than those against Ad5 in populations in North America, South America, sub-Saharan Africa, and Southeast Asia [179] , reducing the potential impact of pre-existing immunity against the vector on the success of this candidate. As of September 2020, the AstraZeneca vaccine is in Phase 3 clinical trials [180] J o u r n a l P r e -p r o o f Journal Pre-proof (ISRCTN89951424, NCT04516746), and a Phase 3 trial for the Janssen candidate has also been registered inducing less host response to the vector than unmodified Ad5 [181] [182] [183] . They evaluated this vector used to deliver the SARS-CoV-2 S protein, as well as the N protein [184] . The justification for inclusion of the nucleoprotein antigen in the encoded sequence was to induce T cell-mediated immunity to N protein epitopes in addition to S protein epitopes. The N protein in this case was coupled with what they call and Enhanced T Cell Stimulation Domain (ETSD). The precise structure of this domain was not disclosed by the group, but they disclose that it is designed to direct the protein to induce endosomal processing and MHC Class II presentation. Other groups have accomplished this task with SARS-CoV N by fusing the N antigen to lysozome-associated membrane protein (LAMP) [185] . In the preclinical evaluation of this platform, they reported the generation of anti-S and N antibodies, neutralizing antibodies, and CD4 and CD8 T cell responses against both S and N peptide pools after subcutaneous prime-boost vaccination of CD1 mice. Efficacy in protection against viral challenge was not reported. A Phase 1 clinical trial of this vaccine candidate is currently underway, using a prime-boost schedule and subcutaneous injection (NCT045917170).

Another adenoviral vector using a simian adenovirus encoding the spike protein is reportedly in development by a collaboration of Reithera, LEUKOCARE, and Univercells. The trial provides for a prime-only vaccination, which is logistically preferable and circumvents the issue of developing vector-specific immunity to inhibit the effectiveness of a boost. However, it is unclear what may be unique to this formulation to allow it to generate significant immunity from a prime-only schedule. LEUKOCARE's contribution to the collaboration will be developing a formulation to stabilize the viral vectors. They have recently published and extensively patented methods using algorithmic methods to identify mixtures of excipients which can stabilize diverse types of biopharmaceuticals. With this method they used Design of Experiments principles combined with accelerated stability testing to determine mixtures of excipients which most effectively preserved the stability of adenoviral vectors [186, 187] . It remains to be seen what degree of stability this method may be able to impart 

Vaccines using vesicular stomatitis virus (VSV) as a vector have been developed for a variety of viral pathogens. This technology was initially developed for application as an influenza vaccine, where genes encoding for influenza HA were inserted into the VSV genome, resulting in a replication-competent vector which induced protective immunity against influenza A challenge in mice after intranasal administration [188] .

This vector was subsequently improved by altering or deleting the VSV glycoprotein gene from the viral sequence to reduce vector-induced pathogenesis and abrogate the generation of anti-vector immunity [189] .

Use of this platform with the VSV glycoprotein deleted, and encoding for the Ebola glycoprotein (rVSV-ZEBOV) was shown to induce protection against an Ebola virus challenge in animal models [190] . Clinical evaluation demonstrated the safety and immunogenicity of this platform, and it was subsequently the first licensed Ebola vaccine and given the brand name Ervebo (Merck) [191, 192] .

Similar efforts were also undertaken to use VSV as a vector for vaccines against various coronaviruses. Intranasal immunization with this vaccine prevented SARS-CoV replication in mice challenged intranasally, and protection was determined to be antibody-mediated [193] . An alternative strategy of fully replacing the VSV glycoprotein with the SARS-CoV S protein, resulting in a replication-incompetent vector, was compared to the replication-competent vector that retained the glycoprotein for use as an intramuscularly-administered vaccine [194] . A greater humoral response against the S protein was observed in mice given the replicationincompetent vector compared to the replication-competent vector.

Two studies described the generation of VSV vectors encoding the S protein of SARS-CoV-2 for use as vaccines. In one study, VSV encoding S protein as well as green fluorescent protein (GFP), and trace amounts of VSV glycoprotein, was produced. This vector was initially developed to provide a tool to identify inhibitors of SARS-CoV-2 S-protein-mediated entry [195] . In a subsequent study, VSV glycoprotein was added to the J o u r n a l P r e -p r o o f Journal Pre-proof vector in order to permit glycoprotein-mediated cell entry and a single round of replication upon administration to mice, as murine Ace2 differs from human [196] . Intranasal vaccination of BALB/c mice using a prime-only or prime-boost regimen induced anti-S titers and neutralizing titers, with a significantly greater overall humoral response from the prime-boost regimen. Mice were transfected with hACE2 and administered an anti-IFNAR1 monoclonal antibody in a disease model which was shown to recapitulate lung pathology associated with human infection [197] . Mice vaccinated with the VSV vector encoding GFP and S protein demonstrated reduced viral lung titers and lung pathology after a SARS-CoV-2 challenge, with the prime-boost receiving mice having significantly lower viral loads and lung pathology.

In another study investigating VSV as a vector to vaccinate against the S protein, a VSV vector initially bearing both S protein and the VSV glycoprotein was used to infect Vero cells [198] . The resulting vector was then serially passaged through Vero cells to increase viral titer and infectivity. As this also resulted in mutations to the vector S protein, the authors verified antigenic similarity of the vector S protein to that of SARS-CoV-2 by comparing the capacity of COVID-19 patient convalescent serum to neutralize their vector and SARS-CoV-2.

They then subcutaneously immunized Syrian golden hamsters with this vector on a prime-only schedule and challenged them with SARS-CoV-2 vector 25 days later. Immunized mice demonstrated significantly lower viral titers, weight loss, and lung tissue pathology relative to unimmunized controls, as well as robust induction of SARS-CoV-2 neutralizing titers. This vaccine, developed by the Israel Institute for Biological Research, is slated to begin clinical trials in Israel in November 2020. Another Phase 1 clinical trial instituted by a collaboration between Merck and the International AIDS Vaccine Initiative (IAVI) using VSV with its glycoprotein replaced by the S protein will be conducted in the United States (NCT04569786).

Themis has reported a measles virus based vaccine that expresses the SARS-CoV-2 S protein [199] .

The measles vector is based on the Schwarz vaccine strain which has 10 amino acids different from the Edmonston B strain, a strain abandoned as a measles vaccine a quarter century ago [200] . Previously,

Themis' platform has shown efficacy in Phase 1 and 2 clinical trials for Chikungunya [201, 202] . In a preclinical evaluation of their SARS-CoV-2 formulation, IFNAR -/-577 -CD46Ge mice were used because these mice can become infected with measles. Soluble SARS-CoV-2 S-protein with alum was used as a control. Using a prime and boost schedule (Day 0 and 28), total antibody concentration was lower on day 28 and 49 than observed Vaxart previously has performed a clinical trial using their technology for the development of a norovirus vaccine [204] . In Vaxart's clinical trial for norovirus, 61% of the patients in the low dose group made a 2-fold increase over the placebo group, while the high dose group had 78% increase in antibodies over the placebo group. Additionally, by delivering an adenovirus orally, they were able to generate IgA antibodies with a 16-fold increase compared to the controls. Based on this data, Vaxart has announced that they are developing a vaccine against SARS-CoV-2 [159] . showed delivery of plasmids effectively to DCs to induce gene expression as well as DC activation [207] .

Symvivo is filing for a Phase 1 clinical trial (NCT04334980) to evaluate this technology further. Based on their website, they are developing a vaccine against the S-protein, nucleocapsid protein and the matrix glycoprotein [208] . The type of bacteria that they are using is not known.

Artificial Antigen Presenting Cells.

Shenzhen Geno-Immune Medical Institute has registered for a Phase 1 clinical trial (NCT04299724)

using an Artificial Antigen Presenting Cell (aAPC) (Figure 4) . aAPCs are a technology that was first reported in 1997 where Vaccinia virus was constructed to express MHC II and a co-stimulatory signal, resulting in antigen specific T cell responses [209] . Since their development MHC and co-stimulatory signals have been added to a variety of additional substrates including those which are protein, polymer, inorganic material and cell based [210] . These platforms have been applied for cancer and infectious disease vaccines as well as to create tolerance [210, 211] . However, in one of the first clinical trials with the technology, the trial was delayed due to the inability to generate GMP quality K562 cells and although cancer co-therapy trials have been performed [212] , there are some concerns about the platform because the K562 is a malignant cancer clone, which is irradiated to halt replication, but nonetheless there are reservations about giving it to cancer patients [213] .

Synthetic aAPCs have been used ex vivo to stimulate CAR T cells in clinical trials but have yet to have clinical trials involving their use [213, 214] .

Some barriers to the development of aAPC in the clinic are likely due to the number of HLA and peptides that would be required in an outbred population, particularly for cancer where immunogenic cancer antigen can be difficult to identify [214] . Indeed, the K562 trial had multiple HLAs prepared for their constructs [213] . For infectious disease vaccines, antigen selection may be easier due to the simplicity of viruses compared to cancer, but HLA matching would still be critical to avoid rejection of the cells and allow them to effectively present antigen. This application then may give aAPCs an application to shine and allow further development of this vaccine technology.

Shenzhen Geno-Immune Medical Institute used lentivirus, NHP/TYF, infect cells and encode various proteins from SARS-CoV-2. It is not clear the exact technology being used with this method; however the leadership of this company, published a methods paper in 2010 on using the NHP/TYF lentivirus to infect DCs for vaccine applications [215] . In their methods paper they describe how unlike other viruses, lentiviral J o u r n a l P r e -p r o o f Journal Pre-proof transduction in DCs can occur with a relatively high efficiency. Prior research has shown that a lentivirus can be used to deliver the gene for IL-12 or siRNA suppressing IL-10 to induce a DC that can promote Th1 responses [216] . The NHP/TYF expresses both a reporter gene, and siRNA, and can transduce human immature DCs by over 90%. Patient's PBMCs are drawn, and then DCs are expanded by culturing the PBMCs with IL-4 and GM-CSF. After a five-day culture, the DCs are transfected with the lentivirus and then can be injected back into the patient. For trials involving this technology, patients will be administered five million aAPCs. There is no published data on how effective this method is on generating an immune response, but it can be hypothesized that this method is highly expensive and would be difficult to apply to vaccinate a large population. From a formulation perspective, a major aspect of COVID-19 vaccine development which still needs to be addressed is use of storage outside of the cold chain, antigens other than S-protein, and alternative routes of vaccination.

Modification of the spike protein to increase its expression has also been shown to increase its stability [88, 217] , and some preliminary results also have shown a degree of thermostability for RBD-encoding mRNA

LNPs [147] . with Moderna also specifying storage at -70 °C in their Phase 2 trial protocol, and expiration of thawed vaccine after 8 hours at room temperature [125] . This is an area where LNP RNA vaccines have a significant logistical disadvantage, although CureVac reports an LNP which has three-month thermostability at 5 ºC [58, 146, 218] .

As noted above, Novavax performed some preliminary stability analysis of its protein nanoparticle formulation on the timescale of 48 hr. Each of these studies have been for only short periods of time and some do not necessarily recapitulate the potential for high humidity and heat conditions that may be seen when shipping vaccines outside of the cold chain. Breakages in the cold chain are common and a major impediment to distribution of currently approved vaccines, while it has been shown that vaccines which can be administered in the 'Controlled Temperature Chain' rather than the cold chain can demonstrate increased rates of vaccine coverage with a lower economic burden [219, 220] . The use of biomaterial delivery vehicles and excipients are currently being explored as means to stabilize biologic drugs and vaccines [221] , and exploring these strategies offers a critical and seemingly underexplored opportunity to increase the effective and equitable distribution of any vaccine which may be approved.

For most vaccine candidates currently in clinical trials, S-protein or regions of S-protein are the main target antigen. This can be attributed to the fact that all potently-neutralizing antibodies recovered from patient convalescent sera have targeted parts of the spike, whether the RBD or elsewhere [222, 223] . However, for future vaccine development, there are other potential targets for protection ( Figure 5 ). For example, a microarray screen of patient convalescent plasma for antibodies against various SARS-CoV-2 proteins exhibited antibodies specific for the N, ORF9b, and NSP5 proteins in addition to S [224] . Although antibodies J o u r n a l P r e -p r o o f Journal Pre-proof against these proteins may be non-neutralizing, it is known that non-neutralizing antibodies can play a role in control of infection against diverse viral pathogens including influenza [225] [226] [227] , HIV [228, 229] , Ebola [230] ,

and Marburg [231] through mechanisms including cooperativity and antibody-dependent cellular cytotoxicity, although they also bear the risk of inducing ADE as detailed above.

Moderbacher et al. demonstrated that prevention of disease generally correlates well with neutralizing antibody levels, while resolution of disease generally correlates well with CD4 T cell response rather than neutralizing antibody titers [232] . Indeed, following infection, there is an association of higher titers with worse disease [233, 234] . Thus, it could be important for a vaccine designed to prevent disease to induce a strong CD4 T cell response, especially in the case that vaccine approval is only contingent on 50% reduction in disease incidence [235, 236] . Kiyotani [239, 240] . The important role of CD4 T cell-mediated immunity in reducing disease severity, and the large number of T cell epitopes present in proteins besides S in SARS-CoV-2, suggest inclusion of these proteins in future vaccine formulations may confer a protective benefit.

An additional consideration in the design of emerging vaccine platforms is the use of adjuvants. A Th1 response with minimal non-neutralizing antibodies are known to mitigate immunotoxicity. PAMP-based adjuvants can promote a strong Th1 response, especially in contrast to Th2 adjuvants like alum, MF59 and AS03 that would fall short. Many of the emerging technologies are non-adjuvanted, which will likely require J o u r n a l P r e -p r o o f Journal Pre-proof multiple boosts to achieve protective immunity. In considering the viral vectors that cannot be easily used multiple times, an adjuvanted subunit vaccine may be an alternative to include as the prime or boost. A similar approach has been seen previously with a non-adjuvanted DNA prime and adenovirus boost as a HIV vaccine (HVTN 505).

Another area where formulations can address major logistical problems is in the need for sterility and trained medical professionals in the administration of vaccines. Many of the formulations mentioned in this review require either intramuscular or intradermal injection using sterile equipment by a trained professional.

Especially given the need for both a prime and a boost injection for most formulations, this constitutes a massive logistical burden to achieve the amount of coverage to achieve herd immunity. Formulations using alternative delivery routes may provide some relief from this burden. This includes orally administered vaccines such as the aforementioned vaccine in development by Vaxart or the intranasal Merck/IAVI VSV vaccine.

Another potential solution to this problem could come in the form of microneedle patches. These patches have been evaluated preclinically and clinically, where they have been shown to stabilize antigen and adjuvant components while safely generating robust immune responses, while having the major advantages of being small, relatively thermostable, and potentially allowing for self-administration [241] [242] [243] . A microneedle formulation for SARS-CoV-2 vaccination has even been evaluated preclinically by Kim et al. [244] , and is proceeding towards clinical evaluation.

In the case of pathogens which infect through a mucosal surface, sterilizing immunity is often associated with mucosal immune responses. It is important to consider the distinction between induction of sterilizing and nonsterilizing immunity. A sterilizing immune response will inhibit viral infection and replication within the host, while a non-sterilizing immune response will permit infection in the host, while still potentially preventing disease. A host with nonsterilizing immunity may become infected by a pathogen and spread it to others despite being asymptomatic. Given the substantial role of asymptomatic carriers in the spread of SARS-CoV-2, induction of sterilizing immunity by a vaccine would be a highly desirable characteristic because it could limit viral transmission in addition to directly preventing disease.

For a respiratory pathogen like SARS-CoV-2, induction of high levels of secretory IgA is considered to have the strongest potential for inducing sterilizing immunity, as this class of antibody is considered to play the J o u r n a l P r e -p r o o f Journal Pre-proof greatest role in protection of the upper respiratory tract where viral laden droplets are most likely to make first contact. In contrast, the lower respiratory tract has a greater proportion of IgG. While natural infection with SARS-CoV-2 has been shown to induce both secretory IgA and IgG, vaccination by injection, especially intramuscular, is often ineffective in inducing secretory IgA. Given that most of the clinical trials mentioned above use intramuscular or intradermal injection, it is possible that sterilizing immunity will not be achieved by many candidates.

For vaccines against respiratory pathogens, the most common method for inducing a mucosal response is through intranasal vaccine administration. For example, Flumist® is an attenuated influenza virus vaccine licensed by the FDA for intranasal administration. Intranasally-administered subunit vaccine formulations can also be used to induce a robust mucosal response, and a number of adjuvants have been developed to increase their immunogenicity [245] [246] . There is thus a compelling case for development of intranasal vaccine formulations against SARS-CoV-2 [247] .

Indeed, a recent study by Hassan et al. demonstrated that a prime-only intranasal immunization with a chimpanzee adenovirus encoding the 2P-stabilized S protein could protect against disease and induce significant S-and RBD-specific levels of serum IgA in an hACE-2 receptor transgenic mouse model. They observed neither infectious SARS-CoV-2 virus nor viral RNA in the lungs of the mice four days after viral challenge, but still observed some viral RNA in the nasal wash and turbinates. This RNA could have been from the original challenge dose rather than viral replication. They investigated anti-N IgM and IgG serum titers eight days after viral challenge, finding no significant difference in titers against this antigen, which is not encoded for by the vaccine vector. This experiment was meant to demonstrate that there was no viral replication and thus no generation of NP against which a significant humoral response could be raised, although it still does not provide definitive proof of sterilizing immunity.

While injected vaccines are conventionally considered incapable of generating a mucosal response [248] , there is some evidence for induction of mucosal immunity to vaccines injected via diverse routes at nonmucosal surfaces. This has occurred using specific adjuvant compounds or unconventional routes of administration [249] [250] [251] [252] . As intranasal immunization can result in reduced levels of systemic antibody induction relative to an intramuscular injection [253] , use of a one of these methods to induce a combination of robust mucosal and systemic immunity through intramuscular injection merits further investigation.

J o u r n a l P r e -p r o o f Journal Pre-proof

Current clinical trial designs and recommendations from the WHO assess vaccine efficacy as the prevention of disease caused by infection of SARS-CoV-2, rather than infection itself (sterilizing immunity).

Thus, these trials will not thoroughly assess the capacity of each vaccine candidate to induce sterilizing immunity or prevent disease transmission. It is thus possible that, even with an effective vaccine, we will not achieve herd immunity, and instead COVID-19 will become a recurrent or seasonal disease [254, 255] . While there are practical limitations on the direct assessment of sterilizing immunity during trials involving community transmission, quantification of S-protein specific and/or virus-neutralizing IgA in serum and/or nasal midturbinates should be feasible and could provide more insight into the potential of each vaccine to limit viral spread [256] . Other strategies involving consistent monitoring of viral load and in-depth contact and cluster tracing may also give some indication of successful prevention of infection and transmission [257] . It was recently announced that the United Kingdom company Open Orphan/hVivo will be initiating human challenge trials with SARS-CoV-2, while the governments of Belgium and the United States have also allocated funding for similar trials. While the ethics and value of such trials are controversial, human challenge trials could provide the potential to evaluate the induction, duration, and correlates of sterilizing immunity against SARS-CoV-2 in humans by administering a known dose of infectious virus at a defined time [258] . Similar insights could also be achieved at a lesser risk to trial subjects through development of a dosing regimen or attenuated virus which does not cause disease [259] .

While comprehensively addressing the details of clinical trial design is beyond the scope of this review, it is critical that Phase 3 trials for each of these vaccines are undertaken in a manner to provide the greatest protection to humanity. Current FDA and WHO guidelines set the necessary protective efficacy as 50%, with a recommendation that this be defined as protection against symptomatic or severe COVID-19 [235, 236] . While this endpoint has been reported by several vaccine candidates [128, 139, 140, 177] , it will still be important to evaluate candidates for more robust endpoints, including duration of immunity, potential for sterilizing immunity, and efficacy in pediatric, older, or immunocompromised individuals. The capacity to achieve these more demanding endpoints may be best assessed through trials which directly compare different candidates, which are being organized by organizations like the WHO [260] . It is also critical to ensure the recruitment of racial and ethnic minority populations in trial recruitment, as these populations have been disproportionately J o u r n a l P r e -p r o o f Journal Pre-proof affected by COVID-19 [261] [262] [263] . The Moderna Phase 3 Trial highlights diversity can be achieved in the study's participants and greater patient diversity across all trials should be sought [129] . Demonstration of efficacy in Phase 3, resulting in approval for distribution, could have deleterious effects on further evaluation of each candidate by slowing recruitment to other efficacy trials. It could also result in unblinding of the trial as the placebo participants could be justified to receive the treatment after it is proven efficacious. This would eliminate the a control group required for effective long-term efficacy and safety monitoring, and so must be carefully considered [264] .

Overall, a wide variety of approaches are being taken to rapidly develop a vaccine against SARS-CoV-2. Due to the need to simply know the sequence of the desired antigen to begin manufacturing, viral and nonviral nucleic acid vaccines seem to be the quickest out of the gate. These plug-and-play platforms are advancing rapidly through clinical trials. Close behind are more traditional inactivated and subunit vaccines, followed by emerging technologies that apply cells or bacteria to generate potential protective responses.

Spike-and RBD-binding, virus-neutralizing antibodies have been successfully raised using a myriad of approaches, and these antibodies have correlated strongly with protection from infection in multiple animal models. In most cases where it has been evaluated, vaccine candidates have been successful in inducing a Th1-skewed T cell response. While the determinants of immune pathology in COVID-19 have not been definitively determined, avoiding a Th2 skew has some theoretical basis for reducing immune pathology.

The overall effect of COVID-19 vaccine development has been a massive invigoration of the field of pandemic vaccine development. It has made real the theoretical promise of platforms which only require an antigen sequence, such as mRNA and vector-based platforms, and massively accelerated their development towards rapid Phase 3 evaluation, on a timeline never seen before for vaccines. However, it is important to note that, despite their rapid manufacturing timeline, these platforms encode for an antigen which was developed over a timeline of many years through basic research on coronavirus biology and protein engineering. Large scale investment and unprecedented mobilization of the research community have generated insight into design, manufacturing, formulation, and deployment of vaccine candidates that may pay dividends in the future when society will need to confront the next inevitable infectious disease outbreak.

Coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus

Severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection

A decade after SARS: strategies for controlling emerging coronaviruses

Middle East respiratory syndrome coronavirus (MERS-CoV): A review, Germs

Worldwide Reduction in MERS Cases and Deaths since

Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study

The trinity of COVID-19: immunity, inflammation and intervention

COVID-19 pathophysiology: A review

Pulmonary embolism and increased levels of d-dimer in patients with coronavirus disease

Hypothesis for potential pathogenesis of SARS-CoV-2 infection-a review of immune changes in patients with viral pneumonia, Emerging microbes & infections

Pathology and pathogenesis of SARS-CoV-2 associated with fatal coronavirus disease

Microvascular disease confers additional risk to COVID-19 infection

A comparative epidemiologic analysis of SARS in Hong Kong

Interactions of coronaviruses with ACE2, angiotensin II, and RAS inhibitors-lessons from available evidence and insights into COVID-19

The pivotal link between ACE2 deficiency and SARS-CoV-2 infection

The spike protein of SARS-CoV--a target for vaccine and therapeutic development

Inactivated SARS-CoV vaccine elicits high titers of spike protein-specific antibodies that block receptor binding and virus entry

SARS CoV subunit vaccine: antibody-mediated neutralisation and enhancement

Characterization of humoral responses in mice immunized with plasmid DNAs encoding SARS-CoV spike gene fragments

Adenoviral expression of a truncated S1 subunit of SARS-CoV spike protein results in specific humoral immune responses against SARS-CoV in rats

SARS vaccines: where are we?

Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein

Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor

Structural and functional basis of SARS-CoV-2 entry by using human ACE2

The receptor binding domain of the viral spike protein is an immunodominant and highly specific target of antibodies in SARS-CoV-2 patients

Human immunopathogenesis of severe acute respiratory syndrome (SARS)

Anti-spike IgG causes severe acute lung injury by skewing macrophage responses during acute SARS-CoV infection

The trinity of COVID-19: immunity, inflammation and intervention

Antibody-dependent enhancement and SARS-CoV-2 vaccines and therapies

Immunization with SARS coronavirus vaccines leads to pulmonary immunopathology on challenge with the SARS virus

A double-inactivated severe acute respiratory syndrome coronavirus vaccine provides incomplete protection in mice and induces increased eosinophilic proinflammatory pulmonary response upon challenge

Vaccine efficacy in senescent mice challenged with recombinant SARS-CoV bearing epidemic and zoonotic spike variants

Clinical features of patients infected with 2019 novel coronavirus in

Loss of Bcl-6-expressing T follicular helper cells and germinal centers in COVID-19

Fundamentals of clinical trials

Clinical trial phases

Draft landscape of COVID-19 candidate vaccines

FDA, Vaccines Licensed for Use in the United States

Influenza Vaccine Manufacturing: Effect of Inactivation, Splitting and Site of Manufacturing. Comparison of Influenza Vaccine Production Processes

Egg-based production of influenza vaccine: 30 years of commercial experience

Influenza vaccine manufacture: keeping up with change

Innate responses induced by whole inactivated virus or subunit influenza vaccines in cultured dendritic cells correlate with immune responses in vivo

Development of an Inactivated Vaccine Candidate

African green monkey kidney (Vero) cells provide an alternative host cell system for influenza A and B viruses

Effect of an Inactivated Vaccine Against SARS-CoV-2 on Safety and Immunogenicity Outcomes: Interim Analysis of 2 Randomized Clinical Trials

Immunogenicity and safety of a SARS-CoV-2 inactivated vaccine in healthy adults aged 18-59 years: report of the randomized, double-blind, and placebo-controlled phase 2 clinical trial

Placebo-Controlled Phase III Clinical Trial to Evaluate the Efficacy and Safety of treating Healthcare Professionals with the Adsorbed COVID-19 (Inactivated) Vaccine Manufactured by Sinovac-PROFISCOV: A structured summary of a study protocol for a randomised controlled trial

Seoul National University Hospital to Start Phase 1/2 Clinical Trial of INOVIO's COVID-19 DNA Vaccine (INO-4800) in South Korea

Inovio reports promising data from its Covid-19 vaccine studies

Efficient Targeting and Activation of Antigen-Presenting Cells In Vivo after Modified mRNA Vaccine Administration in Rhesus Macaques

Inside the Company That's Hot-wiring Vaccine Research in the Race to Combat the Coronavirus

NIH clinical trial of investigational vaccine for COVID-19 begins

SARS-CoV-2 mRNA Vaccine Development Enabled by Prototype Pathogen Preparedness, bioRxiv

Safety and immunogenicity of a mRNA rabies vaccine in healthy adults: an open-label, non-randomised, prospective, first-in-human phase 1 clinical trial

Novavax Identifies Coronavirus Vaccine Candidate; Accelerates Initiation of First-in-Human Trial to Mid

Clover's adjuvant choice for Covid-19 an edge, but comparison with IMV's, Novavax's vaccines limited

Clover Biopharmaceuticals starts Phase I Covid-19 vaccine trial

Adjuvants help vaccines work better

Improvement of Pharmacokinetic Profile of TRAIL via Trimer-Tag Enhances its Antitumor Activity <i>in vivo</i>, Scientific Reports

Chimeric molecules and uses thereof

Queensland's, UQ vaccine scientists report positive results from pre-clinical testing

Advax, a delta inulin microparticle, potentiates in-built adjuvant property of co-administered vaccines

Delta inulin: A novel, immunologically active, stable packing structure comprising β-d-*2→ 1+ poly (fructo-furanosyl) α-d-glucose polymers

Optimizing the utilization of aluminum adjuvants in vaccines: you might just get what you want, npj Vaccines

COVID-19 vaccines: neutralizing antibodies and the alum advantage

Development and evaluation of AS03, an Adjuvant System containing α-tocopherol and squalene in an oil-in-water emulsion, Expert review of vaccines

Development of the CpG adjuvant 1018: a case study

A novel lipid nanoparticle adjuvant significantly enhances B cell and T cell responses to sub-unit vaccine antigens

Antibody responses to crucial functional epitopes as a novel approach to assess immunogenicity of vaccine adjuvants

The requirement of lipids for the formation of immunostimulating complexes (iscoms)

Matrix-M™ adjuvant induces local recruitment, activation and maturation of central immune cells in absence of antigen

Matrix-M™ adjuvant: enhancing immune responses by 'setting the stage'for the antigen

The mechanism of action of MF59-an innately attractive adjuvant formulation

Mechanism of action of licensed vaccine adjuvants

Polysorbates 20 and 80 used in the formulation of protein biotherapeutics: structure and degradation pathways

Structural analysis of full-length SARS-CoV-2 spike protein from an advanced vaccine candidate

RNA modifications modulate activation of innate toll-like receptors

A Universal Design of Betacoronavirus Vaccines against COVID-19, MERS, and SARS

Advax Adjuvant: A Potent and Safe Immunopotentiator Composed of Delta Inulin, Immunopotentiators in Modern Vaccines

Severe Acute Respiratory Syndrome-Associated Coronavirus Vaccines Formulated with Delta Inulin Adjuvants Provide Enhanced Protection while Ameliorating Lung Eosinophilic Immunopathology

Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation

Immunogenicity and structures of a rationally designed prefusion MERS-CoV spike antigen

Development of CpG-adjuvanted stable prefusion SARS-CoV-2 spike antigen as a subunit vaccine against COVID-19, bioRxiv

Formation of protein micelles from amphiphilic membrane proteins

Purified coronavirus spike protein nanoparticles induce coronavirus neutralizing antibodies in mice

Matrix-M adjuvant enhances immunogenicity of both protein-and modified vaccinia virus Ankara-based influenza vaccines in mice

Evaluation of a virosomal H5N1 vaccine formulated with Matrix M adjuvant in a phase I clinical trial

SARS-CoV-2 spike glycoprotein vaccine candidate NVX-CoV2373 elicits immunogenicity in baboons and

The spike glycoprotein of the new coronavirus 2019-nCoV contains a furin-like cleavage site absent in CoV of the same clade

Vaccine instability in the cold chain: mechanisms, analysis and formulation strategies

Vaccine stability study design and analysis to support product licensure

NVX-CoV2373 vaccine protects cynomolgus macaque upper and lower airways against SARS-CoV-2 challenge, bioRxiv

Phase 1-2 Trial of a SARS-CoV-2 Recombinant Spike Protein Nanoparticle Vaccine

Molecular pharming -VLPs made in plants

Vaccine delivery: a matter of size, geometry, kinetics and molecular patterns

Influenza virus-like particles produced by transient expression in Nicotiana benthamiana induce a protective immune response against a lethal viral challenge in mice

The Rise and Rise of Nicotiana benthamiana: A Plant for All Reasons

Immunogenicity and safety of a quadrivalent plant-derived virus like particle influenza vaccine candidate-Two randomized Phase II clinical trials in 18 to 49 and >/=50 years old adults

DNA vaccines for targeting bacterial infections, Expert review of vaccines

RNA-based vaccines

The effect of N/P ratio on the in vitro and in vivo interaction properties of PEGylated poly[2-(dimethylamino)ethyl methacrylate]-based siRNA complexes

Highly efficient in vivo gene transfection by plasmid/PEI complexes coated by anionic PEG derivatives bearing carboxyl groups and RGD peptide

Distribution of DNA vaccines determines their immunogenicity after intramuscular injection in mice

Increased gene expression and inflammatory cell infiltration caused by electroporation are both important for improving the efficacy of DNA vaccines

Safety and immunogenicity of an anti-Middle East respiratory syndrome coronavirus DNA vaccine: a phase 1, open-label, single-arm, dose-escalation trial

Tolerability of intramuscular and intradermal delivery by CELLECTRA((R)) adaptive constant current electroporation device in healthy volunteers

Immunogenicity of a DNA vaccine candidate for COVID-19

Intradermal-delivered DNA vaccine provides anamnestic protection in a rhesus macaque SARS-CoV-2 challenge model, bioRxiv

South Korea's Genexine begins phase I/IIa trials for COVID-19 vaccine

Sung, Clearance of persistent HPV infection and cervical lesion by therapeutic DNA vaccine in CIN3 patients

A Phase II, Prospective, Randomized, Multicenter, Open-Label Study of GX-188E, an HPV DNA Vaccine

Sahin, FLT3 ligand enhances the cancer therapeutic potency of naked RNA vaccines

Soluble Spike DNA vaccine provides long-term protective immunity against SAR-CoV-2 in mice and nonhuman primates

Development of Pyro-Drive Jet Injector With Controllable Jet Pressure

Stable Immune Response Induced by Intradermal DNA Vaccination by a Novel Needleless Pyro-Drive Jet Injector

Sanofi and Translate Bio expand collaboration to develop mRNA vaccines across all infectious disease areas

Inhaled Nanoformulated mRNA Polyplexes for Protein Production in Lung Epithelium

Synthesis, Formulations, and Their Biomedical Applications

Stabilized coronavirus spikes are resistant to conformational changes induced by receptor recognition or proteolysis

An mRNA Vaccine against SARS-CoV-2 -Preliminary Report

Safety and immunogenicity of SARS-CoV-2 mRNA-1273 vaccine in older adults

An mRNA Vaccine against SARS-CoV-2 -Preliminary Report

Moderna's coronavirus vaccine is 94.5% effective, according to company data

Moderna signals diversity of its US COVID-19 vaccine trial cohort

Pandemic influenza vaccine manufacturing process and timeline

Trump Administration Selects Five Coronavirus Vaccine Candidates as Finalists

Moderna Applies for Emergency F.D.A. Approval for Its Coronavirus Vaccine

Zika virus protection by a single low-dose nucleoside-modified mRNA vaccination

Phase 1/2 Study to Describe the Safety and Immunogenicity of a COVID-19 RNA Vaccine Candidate (BNT162b1)

A recombinant receptorbinding domain of MERS-CoV in trimeric form protects human dipeptidyl peptidase 4 (hDPP4) transgenic mice from MERS-CoV infection

Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability

Safety and immunogenicity of two RNA-based Covid-19 vaccine candidates

Pfizer and BioNTech Announce Vaccine Candidate Against COVID-19 Achieved Success in First Interim Analysis from Phase 3 Study

New Pfizer Results: Coronavirus Vaccine Is Safe and 95% Effective

Covid-19: Pfizer/BioNTech vaccine judged safe for use in UK

Rabies pre-exposure prophylaxis using intradermal human diploid cell vaccine: immunologic efficacy and cost-effectiveness in a university medical center and a review of selected literature

CureVac´s Optimized mRNA Platform Provides Positive Pre-Clinical Results at Low Dose for Coronavirus Vaccine Candidate

Phase 1 Assessment of the Safety and Immunogenicity of an mRNA-Lipid Nanoparticle Vaccine Candidate Against SARS-CoV-2 in Human Volunteers, medRxiv

Unmodified mRNA in LNPs constitutes a competitive technology for prophylactic vaccines, npj Vaccines

CureVac's COVID-19 Vaccine Candidate, CVnCoV, Suitable for Standard Fridge Temperature Logistics

Rapid adaptation of SARS-CoV-2 in BALB/c mice: Novel mouse model for vaccine efficacy, bioRxiv

Lab to Sidestep Drug Industry to Sell Potential Virus Vaccine

Cationic HDL mimetics enhance in vivo delivery of self-replicating mRNA

Lipids and lipid nanoparticle formulations for delivery of nucleic acids

Self-amplifying RNA SARS-CoV-2 lipid nanoparticle vaccine induces equivalent preclinical antibody titers and viral neutralization to recovered COVID-19 patients

Arcturus Reports Additional Supportive Preclinical Data for its COVID-19 Vaccine Candidate (LUNAR-COV19

Systemic delivery of factor IX messenger RNA for protein replacement therapy

Nucleic acid vaccine composition comprising a lipid formulation, and method of increasing the potency of nucleic acid vaccines

Ionizable cationic lipid for RNA delivery

UNA oligomers for therapeutics, Google Patents

Systemic and mucosal immune responses following oral adenoviral delivery of influenza vaccine to the human intestine by radio controlled capsule

Vaxart Announces Selection of its Oral COVID-19 Vaccine Lead Candidate

Safety, tolerability, and immunogenicity of a recombinant adenovirus type-5 vectored COVID-19 vaccine: a dose-escalation, open-label, nonrandomised, first-in-human trial

CanSino publishes first COVID-19 vaccine data to muted response

Merck, one of Big Pharma's biggest players, reveals its COVID-19 vaccine and therapy plans

Sanofi Expands COVID-19 Pipeline with Translate Bio Partnership

An Inquiry Into the Causes and Effects of the Variolae Vaccinae

Combination attenuation offers strategy for live attenuated coronavirus vaccines

Attenuated SARS-CoV-2 variants with deletions at the S1/S2 junction, Emerging microbes & infections

Pathogenicity, immunogenicity, and protective ability of an attenuated SARS-CoV-2 variant

Mechanism of virus attenuation by codon pair deoptimization

Salmonella Vaccines: Conduits for Protective Antigens

Cell-Based Flu Vaccines

Safety and immunogenicity of a recombinant adenovirus type-5 vector-based Ebola vaccine in healthy adults in Sierra Leone: a single-centre, randomised, double-blind

Phase 1 Trials of rVSV Ebola Vaccine in Africa and Europe

Safety, tolerability, and immunogenicity of a recombinant adenovirus type-5 vectored COVID-19 vaccine: a dose-escalation, open-label, non-randomised

Immunogenicity and safety of a recombinant adenovirus type-5-vectored COVID-19 vaccine in healthy adults aged 18 years or older: a randomised, double-blind

AstraZeneca advances response to global COVID-19 challenge as it receives first commitments for Oxford's potential new vaccine

The Lancet

Why Oxford's positive COVID vaccine results are puzzling scientists

Janssen Vaccine Technology

International seroepidemiology of adenovirus serotypes 5, 26, 35, and 48 in pediatric and adult populations

Coronavirus (Covid-19) vaccine status check: Oxford vaccine most advanced, says WHO

A preliminary and comparative evaluation of a novel Ad5 [E1-, E2b-] recombinant-based vaccine used to induce cell mediated immune responses

Anti-tumor immunotherapy despite immunity to adenovirus using a novel adenoviral vector Ad5

Optimization of vaccine responses with an E1, E2b and E3-deleted Ad5 vector circumvents pre-existing antivector immunity

A Next Generation Bivalent Human Ad5 COVID-19 Vaccine Delivering Both Spike and Nucleocapsid Antigens Elicits Th1 Dominant CD4+, CD8+ T-cell and Neutralizing Antibody Responses, bioRxiv

Immune responses to T-cell epitopes of SARS CoV-N protein are enhanced by N immunization with a chimera of lysosome-associated membrane protein

Effective Stabilization of Viral Vectors in Liquid Using an Algorithm-Based Development Approach

Algorithm-Based Liquid Formulation Development Including a DoE Concept Predicts Long-Term Viral Vector Stability

Vaccination with a recombinant vesicular stomatitis virus expressing an influenza virus hemagglutinin provides complete protection from influenza virus challenge

Attenuated vesicular stomatitis viruses as vaccine vectors

Live attenuated recombinant vaccine protects nonhuman primates against Ebola and Marburg viruses

A recombinant vesicular stomatitis virus Ebola vaccine

The vesicular stomatitis virus-based Ebola virus vaccine: From concept to clinical trials

Long-term protection from SARS coronavirus infection conferred by a single immunization with an attenuated VSV-based vaccine

SARS vaccine based on a replication-defective recombinant vesicular stomatitis virus is more potent than one based on a replication-competent vector

Neutralizing antibody and soluble ACE2 inhibition of a replication-competent VSV-SARS-CoV-2 and a clinical isolate of SARS

Replication-competent vesicular stomatitis virus vaccine vector protects against SARS-CoV-2-mediated pathogenesis in mice

A SARS-CoV-2 infection model in mice demonstrates protection by neutralizing antibodies

A single dose of recombinant VSV-∆ G-spike vaccine provides protection against SARS-CoV-2 challenge, bioRxiv

A Highly Immunogenic Measles Virus-based Th1-biased COVID-19 Vaccine, bioRxiv

A molecularly cloned Schwarz strain of measles virus vaccine induces strong immune responses in macaques and transgenic mice

Immunogenicity, safety, and tolerability of a recombinant measles-virus-based chikungunya vaccine: a randomised, double-blind, placebo-controlled, active-comparator, first-in-man trial

Immunogenicity, safety, and tolerability of the measles-vectored chikungunya virus vaccine MV-CHIK: a double-blind, randomised, placebo-controlled and active-controlled phase 2 trial

Eudragit a versatile polymer: A review

Safety and immunogenicity of an oral tablet norovirus vaccine, a phase I randomized, placebo-controlled trial

Vaxart's COVID-19 Vaccine Selected for the U.S. Government's Operation Warp Speed

Live Bacterial Vectors-A Promising DNA Vaccine Delivery System

Recombinant invasive Lactococcus lactis can transfer DNA vaccines either directly to dendritic cells or across an epithelial cell monolayer

Artificial antigen-presenting cells engineered by recombinant vaccinia viruses expressing antigen, MHC class II, and costimulatory molecules elicit proliferation of CD4+ lymphocytes in vitro

Bioengineering of Artificial Antigen Presenting Cells and Lymphoid Organs

Biomimetic tolerogenic artificial antigen presenting cells for regulatory T cell induction

Long-lived antitumor CD8+ lymphocytes for adoptive therapy generated using an artificial antigenpresenting cell

The Basics of Artificial Antigen Presenting Cells in T Cell-Based Cancer Immunotherapies

One-step artificial antigen presenting cell-based vaccines induce potent effector CD8 T cell responses

Lentiviral vector transduction of dendritic cells for novel vaccine strategies

Alteration of T cell immunity by lentiviral transduction of human monocyte-derived dendritic cells

Structure-based design of prefusion-stabilized SARS-CoV-2 spikes

A thermostable messenger RNA based vaccine against rabies

Hepatitis B vaccination of newborn infants in rural China: evaluation of a village-based, out-of-cold-chain delivery strategy

Economic benefits of keeping vaccines at ambient temperature during mass vaccination: the case of meningitis A vaccine in Chad

Vaccine instability in the cold chain: mechanisms, analysis and formulation strategies

Potent neutralizing antibodies against multiple epitopes on SARS-CoV-2 spike

SARS-CoV-2 neutralizing antibody structures inform therapeutic strategies

SARS-CoV-2 proteome microarray for global profiling of COVID-19 specific IgG and IgM responses

Both neutralizing and non-neutralizing human H7N9 influenza vaccine-induced monoclonal antibodies confer protection

Broadly-reactive neutralizing and nonneutralizing antibodies directed against the H7 influenza virus hemagglutinin reveal divergent mechanisms of protection

Broadly neutralizing anti-influenza antibodies require Fc receptor engagement for in vivo protection

Non-neutralizing antibodies alter the course of HIV-1 infection in vivo

New paradigms for functional HIV-specific non-neutralizing antibodies

Cooperativity enables non-neutralizing antibodies to neutralize ebolavirus

Microbe, Non-neutralizing Antibodies from a Marburg Infection Survivor Mediate Protection by Fc-Effector Functions and by Enhancing Efficacy of Other Antibodies

Antigen-specific adaptive immunity to SARS-CoV-2 in acute COVID-19 and associations with age and disease severity

SARS-CoV-2 infection severity is linked to superior humoral immunity against the spike, bioRxiv

SARS-CoV-2 neutralizing antibody responses are more robust in patients with severe disease, Emerging microbes & infections

Organization, WHO target product profiles for COVID-19 vaccines, Version

Administration, Development and licensure of vaccines to prevent Covid-19: guidance for industry

Bioinformatic prediction of potential T cell epitopes for SARS-Cov-2

Targets of T Cell Responses to SARS-CoV-2 Coronavirus in Humans with COVID-19 Disease and Unexposed Individuals

Potential T-cell and B-cell Epitopes of 2019-nCoV, bioRxiv

A Sequence Homology and Bioinformatic Approach Can Predict Candidate Targets for Immune Responses to SARS-CoV-2

Formulation and coating of microneedles with inactivated influenza virus to improve vaccine stability and immunogenicity

Microneedle-based vaccines, Vaccines for Pandemic Influenza

Safety and efficacy of a novel microneedle device for dose sparing intradermal influenza vaccination in healthy adults

Microneedle array delivered recombinant coronavirus vaccines: Immunogenicity and rapid translational development

Innate Immunity-Based Mucosal Modulators and Adjuvants, Mucosal Vaccines

Toxin-Based Modulators for Regulation of Mucosal Immune Responses, Mucosal Vaccines

As Plain as the Nose on Your Face: The Case for A Nasal (Mucosal) Route of Vaccine Administration for Covid-19 Disease Prevention

SARS-CoV-2 vaccines in development

Stimulation of local antibody production: parenteral or mucosal vaccination?

Nonmucosal alphavirus vaccination stimulates a mucosal inductive environment in the peripheral draining lymph node

Parenteral vaccination can be an effective means of inducing protective mucosal responses

Induction of mucosal immunity through systemic immunization: phantom or reality?

Cold-adapted live influenza vaccine versus inactivated vaccine: systemic vaccine reactions, local and systemic antibody response, and vaccine efficacy: a meta-analysis

What can we expect from first-generation COVID-19 vaccines?

COVID-19 and the Path to Immunity

A simple and reproducible method for collecting nasal secretions in frail elderly adults, for measurement of virus-specific IgA

Understanding COVID-19 vaccine efficacy

Ethics of controlled human infection to address COVID-19

Accelerating development of SARS-CoV-2 vaccinesthe role for controlled human infection models

COVID-19 vaccine trials should seek worthwhile efficacy

Racial and ethnic disparities among COVID-19 cases in workplace outbreaks by industry sector-Utah

Racial and/or Ethnic and Socioeconomic Disparities of SARS-CoV-2 Infection Among Children

Control, Prevention, COVID-19 hospitalization and death by race/ethnicity

Emergency Use Authorization of Covid Vaccines-Safety and Efficacy Follow-up Considerations

Images were in part generated by BioRender.