COVID Vaccines

Although the vaccines currently approved to prevent the infection caused by the severe acute respiratory syndrome coronavirus 2(SARSCoV-2) —known as coronavirus disease 2019 (COVID-19) — and emerging variants, have shown to greatly reduce the rates of serious illness and death, they appear not fully effective at preventing the infection and disease, and long COVID can arise, even after a mild or asymptomatic coronavirus infection. Indeed, as showed by SARS-CoV-2 infection surveillance and vaccination registry data, numerous fully vaccinated people exposed to SARS CoV-2 developed COVID-19. These cases are called vaccine breakthrough infections, although vaccinated people develop symptoms that tend to be less severe than those experienced by unvaccinated people. In any case, people with vaccine breakthrough infections can be contagious and the virus can spread from them to other people.

A recent study shows that, despite a prior infection can protect against SARSCoV-2 reinfections, emerging variants can infect and/or reinfect both previously naturally infected and vaccinated individuals.

Consequently, even if the general opinion that mass vaccination against SARS-CoV-2 contributes to eliminate the transmission of the virus, the world is not yet positioned to end the pandemic emergency.

Among the several causes affecting the vaccine effectiveness, there are:

• mutations in virus sequences, that lead to mutated clusters in the structural proteins;
• reduced protective effectiveness in older age groups;
• efficacy differences, related both to population or geography and to locally circulating variants.

Consequently, the long-lasting protection against SARS-CoV-2 infection, through specific antibody and T-cell responses elicited by the current vaccination or natural infection, represents a global concern.

A comparative analysis recently performed by Fiolet T. et al., shows that all vaccines represent safe and effective tools to prevent severe COVID-19, hospitalisation, and death against all variants of concern, even if the quality of evidence greatly varies, depending on the vaccines considered. However, the Authors claim that the main questions remain the need of a booster dose, the waning immunity, and the not last long duration of immunity.

Moreover, the current vaccines appear not able to elicit an oral mucosal immunity, thus failing in limiting virus acquisition upon its entry through this route. In fact, the induction of mucosal front-line immunity has the potential to mitigate current and future respiratory virus epidemics and pandemics. In addition, since mucosal immunity maximises the individual protection against breakthrough infections, it contributes to decrease the disease severity and the risk for virus transmission upon infections.

Altogether, the immune response elicited by current COVID-19 vaccines, including both T-cell responses and neutralising antibody production, could be improved, within certain limits, by optimising the primary antigen sequence, the doses, the adjuvants, the immunisation regimes, the manufacturing processes, etc.

However, in my opinion, the main problem consists in the particular conformation of the protein S selected, as protective antigen, both in live or inert, attenuated immunogens, or as protein translated from mRNA-based vaccines.

As above discussed, the S protein shows a high binding affinity both to the angiotens in converting enzyme 2 (ACE2) receptor and to soluble ACE2.

The ACE2 is a multifunctional protein that plays a crucial role in several mechanisms, including:

• regulation of renin-angiotensin, kinin-kallikrein systems, and amyloid peptides;
• transport of neutral amino acids;
• amino acid homeostasis regulation;
• interaction with integrins.

Anyway, one of the main roles of ACE2 is to bind and inactive the potent vasopressine peptide angiotensin II (Ang II) by removing the C-terminal phenylalanine residue to yield Ang 1–7.This conversion inactivates the vasoconstrictive action of Ang II and yields a peptide that acts as a vasodilatory molecule.

According to antigen-antibody interaction theory, it is known that antibodies recognise antigens based on their structure as well as content.

At the same way, in biological systems, in addition to antibodies targeting antigens, all biomolecules, including enzymes catalysing their substrates, regulatory proteins binding DNA, and receptor-ligand complex systems, specifically recognise each other through the so-called mechanism “lock and key”. The ligand recognition can be very specific, allowing the binding to only a small part of an antigen or ligand (known as the epitope), and discriminating between highly similar epitopes.

In natural infections, when an immunocompetent animal is exposed to a T-dependent microbial antigen, it will develop an array of antibodies that eachbind to a separate epitope of the antigen.

At the same way, the complex immune sensory system is able to discriminate self- from non-self-antigens, and autoimmune diseases represent the result of the breakdown in any of the mechanisms that maintain unresponsiveness to self (a state known as self-tolerance).

At light of these considerations, since that both S protein of SARS-Cov-2 and the human protein Ang-II, are capable of selectively recognising and binding the ACE 2 receptor, it can be supposed that S protein shares any sequence identity region with Ang-II.

In addition, S protein has been shown to share sequences similar to alveolar lung surfactant proteins, and this molecular mimicry can represent one of the main mechanisms of SARS-Cov-2 infection responsible for inducing the production of self-reactive antibodies in infected host Ref. Letter from Editor: On the molecular determinants of the SARSCov 2 attack - Clinical Immunology 215 (2020) - 108426.

Consequently In any case, the limited immunogenic properties of the S protein used in COVID vaccines could be mainly due to any conformational similarity to Ang-II, and this similarity could explain both the numerous cases of infection occurring among the regularly vaccinated people and the number dramatically increasing of subjects who have  been infected from SARS-Cov-2 for a second time.

This hypothesis can be easily tested by analysing, in a detailed and comparative manner, the conformational dynamics and the protein sequence alignment, to identify specific regions of similarity between S protein, Ang-II and other endogenous components, including surfactant proteins.

Altogether, these considerations should address the current studies both to further investigate the molecular characteristics of S protein potentially associated to its low immunogenicity and to identify other immunogenic SARSCoV-2 epitopes.

The reverse vaccinology approach allowed to select a further list of SARSCoV- 2 antigens as promising candidates for vaccine development.

Among these, there are the virus nucleocapsid components and the membranegly coprotein, that have been reported to exhibit both pathogenic and immunogenic properties.

In particular, membrane glycoprotein, which is the most abundant viral protein in SARS-CoV-2 and plays a crucial role in viral pathogenesis, has been reported to have a highly immunogenic domain, mainly consisting in its C-terminal region.

In addition, specific non-structural polyproteins (NSP), including NSP3, NSP4, and NSP6, involved in the viral adhesion and host invasion, appear to exhibit high protective immunogenicity. Currently, NSPs represent one of the most promising alternative COVID-19 vaccine candidates, since they have already been successfully used to induce protective immunity against other pathogens, including flaviviruses, hepatitis C virus, and HIV-1.

Peptide-based phage display technology can represent an inexpensive and versatile tool for large-scale screening methodology for the identification of potential vaccine candidates against SARS-Cov-2 as well as other microbial infections.

The ability to produce combinatorial peptide libraries with a highly diverse pool of randomised ligands, allows screening and selecting, through an affinity selection-based strategy called “biopanning”, a wide variety of targets with high antigenicity and immunogenicity. Also, peptide libraries can be panned against the antiserum of convalescent individuals, in order to identify both additional protective virus antigens and novel peptidomimetics of pathogen-related epitopes. Combined with bioinformatic approaches capable of identifying immunogenic epitopes, this strategy provides a promising framework for developing a more effective SARS-Cov-2 vaccine.

In addition, the phage-based vaccines can represent a valid strategy for vaccine design, due to being highly stable under harsh environmental conditions, and potent adjuvant capacities.

Phage display vaccines are obtained by expressing multiple copies of an antigen on the surface of immunogenic phage particles, thereby eliciting a powerful and effective immune response.

Recent advances consider the possibility of producing a peptide-directed phage particle that can be administered in an aerosolised form by inhalation. A combinatorial approach for liganddirected pulmonary delivery as a unique route for systemic targeting in vaccination showed to elicit, both in mice and nonhuman primates, a systemic and specific humoral response.

It is also hoped that efforts towards the development of mucosal vaccines consisting in peptide-directed phage particles leading to secretory IgA antibody production can provide a very strong first line of defense, by preventing the virus entry into the mucosa. It is becoming increasingly clear that local mucosal immune responses, that include, in addition to IgA antibodies, local mucosal IgG production, and cytotoxic T lymphocyte activation, are very important for protection against COVID-19 disease. However, one of the main challenges in designing phage  display vaccines consists into assure that, following the insertion of the different SARS-CoV-2 antigen epitopes into the phage particles, the structure of these peptides is maintained intact as in the original three-dimensional conformation. Therefore, mucosal COVID-19 vaccines represent a promise and challenge, mainly due to the needle-free administration, and the ability to induce both mucosal (IgA) and circulating (IgG and IgA) antibodies, as well as T-cell responses. Mucosal immune responses could also contribute to reduce the frequency of asymptomatic SARS-CoV-2 positive individuals, who represent an important factor in triggering and sustaining infection chains.

In conclusion, the most efficient strategy to combat the current pandemic COVID-19 and save millions of human lives worldwide remains active immunisation. To date, current vaccines have been shown to reduce both mortality and the incidence of severe COVID19. Since, there are still some aspects, concerning their efficacy, immunogenicity and safety to be focused, the future studies will have toanalyse and comparegeneticvariants andmutations, and evaluate the conformational similarity degree between the viral S proteins and the human ACE2 receptor and other endogenous proteins, in addition to identify additional immunogenic epitopes and develop other alternative vaccines.

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