When administered alone, protein sub-unit vaccines are immunologically weak.

Their poor immunogenicity may be due to an inability to properly stimulate pattern

recognition receptors, premature degradation of the proteins, or incomplete post-

translational modifications [9]. Therefore, they require the addition of an adjuvant to

enhance the immune response. Adjuvants are immunostimulatory molecules that work

by activating pathways in the innate immune system that recognize pathogens and

danger signals [61]. Different adjuvants work by activating different receptors, which

eventually leads to downstream activation of the innate immune system and subse-

quently the adaptive immune system [61]. The choice of adjuvant is very important in

that it can also skew the T-cell response towards Th1 or Th2 response, which as dis-

cussed previously, is very important regarding vaccine efficacy and avoiding adverse

effects such as ADE [9]. The use of an adjuvant also allows a reduction in the antigen-

dose required to elicit an immune response. Once injected, protein vaccines are taken

up by dendritic cells and the antigens are subsequently presented on MHC class I and II

molecules, which activates CD8+ and CD4+ T-cells and B-cells [26].

Protein sub-unit vaccines can also be delivered within or conjugated to nano-

particle carrier molecules to increase their immunogenicity and decrease their de-

gradation. The carriers can be lipid, polymeric, or metal based. Furthermore, the

carriers can be designed to specifically target certain cell-types [62]. When using

nanoparticles, it can be advantageous to encapsulate the antigen and adjuvant together

thus ensuring synchronous delivery to the same antigen-presenting cell [18]. This

can help reduce any off-target side-effects due to the adjuvant. Furthermore, non-

synchronous delivery of the antigen and adjuvant can result in activation of immune

system against host proteins rather than the antigen leading to autoimmunity [18].

Protein vaccines can generally be divided into two categories, protein sub-units

and virus-like particles (VLPs). VLPs are empty virus shells that, unlike viral

vectors, contain no genetic material. For more details on VLPs, see Chapter 10.

They can be produced by expressing the viral structural genes in an in-vitro ex-

pression system resulting in the self-assembly of the viral skeleton. VLPs can also

be made by chemically linking antigenic proteins to blank VLP templates [26].

Because VLPs present the antigen in a 3-D conformation similar to the native

pathogen, they may be immunogenic enough to not require the addition of an ad-

juvant [63]. Notable VLP vaccines currently used include Engerix (hepatitis B),

Rocombivax (hepatitis B), Cervarix (HPV), and Gardasil (HPV) [17,26].

Contrary to whole virus vaccines, one disadvantage of protein sub-unit vaccines

is their reliance on a single antigen. When a virus is grown under the selective

pressure of a single monoclonal antibody, any mutations in the viral protein can

lead to loss of vaccine efficacy or complete escape [64].

There are several protein sub-units and VLP vaccines being developed against

SARS-CoV-2, most of which target the S-protein. Several have already made it to

Phase 3 trials and are approved for use. One example is the vaccine produced by

Novavax known as NVX-CoV2373. The vaccine is composed of a recombinant

trimeric, full-length S-protein in the pre-fusion conformation state. The vaccine is

produced using an engineered baculovirus that contains the gene encoding the S-

protein. Insect cells are infected with the baculovirus resulting in their expression of

the S-protein trimers, which are subsequently extracted and purified. Polysorbate 80

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