KATHMANDU UNIVERSITY

DHULIKHEL, KAVRE

Vaccinees against emerging diseases SARs and MERS

(Vaccine Production)

Submitted to:

Mrs Ashna Dhakal (Lecturer)

Department Of Biotechnology

Kathmandu University

Submitted By:

Sudarshan Lamichanne (14)

Amar Khatri Chettri (12)
Raj Prateek Rai (24)
4th year

Date of Submission: September 7, 2020

Abstract

The emergence of the severe acute respiratory syndrome (SARS) that resulted in a pandemic in
2003 and MERS in 2012 spurred a flurry of interest in the development of vaccines to prevent
and treat the potentially deadly viral infection. Researchers around the world pooled their
scientific resources and shared early data in an unprecedented manner in light of the impending
public health crisis. There are still large gaps in knowledge about the pathogenesis of the corona
viruses. While significant advances have been made in the development of animal models, the
practicality of their use may be hampered by a lack of pathological similarity with human
disease. Described here are issues related to progress in vaccine development and the obstacles
that lie ahead for both researchers and regulatory agencies.
The Vaccine design process

Among the3 various classes of beta corona virus that infect humans, notably three beta-
CoVs (SARS-CoV, MERS-CoV, andSARS-CoV-2) are life-threatening. Till date, no clinically
effective prophylactics or therapeutics are available for the prevention or treatment which
creates the urgency for the development of a vaccine against these infective viruses.

In general 3 major classes of SARS vaccines are under development:

1) Inactivated SARS-CoV

Figure- SARS-CoV virus structure

(credits - http://www.who.int/csr/sars/en)

• This virus expresses several structural proteins, including nucleocapsid (N),

membrane (M), envelope (E), and spike (S). All may serve as antigens to induce
neutralizing antibodies and protective responses. In general, prior to identification
of the protein that contains the major neutralizing epitopes, the inactivated virus
may be used as the first-generation vaccine because it is easy to generate whole
killed virus particles. Studies and reports have shown that Corona viruses
inactivated with formaldehyde, UV light, and β-propiolactone can induce virus-
neutralizing antibodies in immunized animals.
But this approach is partly risky and isn’t specific. Production workers are at risk for
infection during handling of concentrated live SARS-CoV, incomplete virus
inactivation may cause SARS outbreaks among the vaccinated or unvaccinated
populations. Also immune suppressed people are under great threat if any case an
outbreak occurs. Once the neutralizing epitopes are identified, the inactivated virus
 vaccine should be replaced by vaccines based on fragments containing neutralizing
epitopes since they are safer and more effective

2) Full-length S protein

Figure- S protein moeclular view

Credits - Yang, Y., Xiao, Z., Ye, K. et al. SARS-CoV-2: characteristics and current advances in research. Virol
J 17, 117 (2020). https://doi.org/10.1186/s12985-020-01369-z

The S protein of SARS-CoV, a type transmembrane glycoprotein, is responsible for

virus binding, fusion, and entry and is a major inducer of neutralizing antibodies. S
protein consists of a signal peptide and 3 domains: an extracellular domain, a
transmembrane domain, and an intracellular domain. Its extracellular domain
consists of 2 subunits, S1 and S2.
The S1 subunit is responsible for virus binding to the receptor, angiotensin-
converting enzyme 2 (ACE2). A fragment located in the middle region of the S1
subunit is the receptor-binding domain (RBD) for ACE2.
 credit- https://www.mdpi.com/books/pd
fview/book/1893

After the signal peptide, the extracellular domain consists of 2 subunits, S1 and S2.
The S1 subunit is responsible for virus binding to the receptor, angiotensin-
converting enzyme 2 (ACE2) for SARS- cov whereas, MERS-CoV uses human CD26
(also known as human dipeptidyl peptidase 4)
Infection by SARS-CoV is initiated by binding of RBD in the viral S protein S1 subunit
to ACE2 on target cells.  The S2 subunit, which contains a putative fusion peptide
and 2 heptad repeats (HR1 and HR2), is responsible for fusion between the viral and
target cell membranes. Infection by SARS-CoV is initiated by binding of RBD in the
viral S protein S1 subunit to ACE2 on target cells. This forms a fusogenic core
between the HR1 and HR2 regions in the S2 domain that brings the viral and target
cell membranes into close proximity, which results in virus fusion and entry.
All of this scenario indicates that the S protein may be used as a vaccine to induce
antibodies for blocking virus binding and fusion.

Several recombinant vector-based vaccines expressing SARS-CoV S protein have

been assessed in preclinical studies.Studies from Chinese microbiologists Yang , Kong
and Huang reported that a candidate DNA vaccine encoding the full-length S protein
induced neutralizing antibodies and protected mice from SARS-CoV. Using DNA
vaccines encoding the full-length and segments of S protein to immunize rabbits,
higher titers of neutralizing antibodies were produced and was demonstrated that
major and minor neutralizing epitopes are located in the S1 and S2 subunits,
respectively .
Reports have also shown that intranasal or intramuscular inoculations of mice with
highly attenuated modified virus Ankara (MVA) vaccines encoding full-length SARS-
CoV S protein also produce neutralizing antibodies. Studies in African green
monkeys reported that mucosal immunization with an attenuated parainfluenza
virus expressing S protein resulted in production of neutralizing antibodies and
protected animals from infection with SARS-CoV. 
 These are generally kind of a Viral-vector based vaccines which usesa viral
backbone to introduce a SARS-CoV-2 gene into the host. This strategy can enhance
immunogenicity without an adjuvant and promotes a robust cytotoxic T cell
response to eliminate virus-infected cells.

All of these data suggest that the S protein can induce neutralizing antibodies and
protective responses in immunized animals

3) Those based on fragments containing neutralizing epitopes

These include SP - signal peptide; RBD - receptor binding domain; FP - fusion

peptide; HR - heptad repeat; TM - transmembrane domain; CP -cytoplasm domain.
IDS - immunodominant sites I to V.

So far, most of potent neutralizing monoclonal anti-bodies are against CoV RBD.
RBD contains the major neutralizing epitopes in the S protein. The recombinant RBD
may be used as an efficacious and safe vaccine for preventing infection by SARS-CoV
strains with distinct genotypes. Therefore, RBD is an attractive vaccine target
because it can focus the immune response on interferenceof receptor binding
because unlike full-length S protein, RBD does not contain immunodominant sites
that induce nonneutralizing antibodies. RBD sequences are relatively conserved.
Thus, recombinant RBD or vectors encoding RBD may be used as safe and
efficacious vaccines. To date, a number of RBD-basedvaccines have been reported in
development against MERS and SARS

However, RBD-based subunit vaccines may face some important challenges, mostly
arising from their relatively low immunogenicity, which must be combined with
appropriate adjuvants.
A Universal Design of Betacoronavirus Vaccines against COVID-19, MERS, and SARS

A proposed tested model of a vaccine design as suggested by researchers of Beijing

Institutes of Life Science in Guangzhou , China describes a universal design of beta-
CoV immunogen that overcomes the immunogenicity limitation of RBD-based
vaccine.

The process can be sumarized as follows:

1. Identification of a Dimeric Form of MERS-CoV RBD as a Superior Immunogen

2. Validation of Vaccine Protection for MERS-CoV RBD-Dimer In Vivo

3. Structural Characterization of MERS-CoV RBD-Dimer

4. Structure-Guided Design of the RBD-SC-Dimer

Figure- Dimer S protein incubation in immunized mice

credits- Yang, Y., Xiao, Z., Ye, K.  et al.  SARS-CoV-2: characteristics and current advances in research.  Virol J 17, 117 (2020).
https://doi.org/10.1186/s12985-020-01369-z
What they found is that, after assessing the immunogenivcity of the RBD dimer and
monomer, A disulfide-linked dimeric form of MERS-CoV RBD significantly enhanced
the antibody response and neutralizing anti-body (NAb) titer compared to the
conventional monomeric form.

In a mouse model, it conferred protection against MERS-CoV infection. To further

explore the protective efficacy of RBD-dimerin vivo,the immunized mice were
transduced via the intranasal (i.n.)route with adenovirus expressing hCD26 as the
MERS-CoV-sensitive animal model. Five days later, the transduced mice were
intranasally challenged with some amount of plaque-forming unit (PFU) of MERS-
CoV. At 3 days after challenge, lung tissues were harvested for virus titer detection
and pathological examination. Consistent with the high serological NAb titers in
RBD-dimer-vaccinated mice, virus loads were reduced ~100- to 1,000-fold in lung
compared to the other monomer studies

Crystal structure revealedthe RBD-dimer fully exposed dual receptor binding

motifs(RBMs), the major site recognized by Nabs. To increase dimerstability, the
immunogen was further engineered as a versionof tandem repeat single chain dimer
(sc-dimer) by structure-guided design without introducing any exogenous
sequence. The RBD-sc-dimer retained high vaccine efficacy as the disulfide-linked
dimer. This strategy was further generalized to develop vaccines against the other
two highly pathogenicbeta-CoVs, SARS-CoV-2 and SARS-CoV. Notably, RBD-sc-dimer
design significantly increased the immunogenicity and enhanced NAb titers
between 10- to 100-fold compared to the conventional RBD-monomer, indicating its
feasibility as a universal strategy for beta-CoV vaccine design. The RBD-sc-dimers of
MERS-CoV and SARS-CoV-2 can be further developed for pilot scale production in
GMP grade manufacturing. Both can be produced at a high yields in an industry-
standard cell system. This suggests its scalability and promise for further clinical
development to control SARS, MERS and the ongoing COVID-19 pandemic.
Ethical considerations

For the process of human challenge studies, there are a lot of ethical factors that need to be considered.
Some of the factors are explained below:

1. Public health benefits

The ethical acceptability of human challenge studies would be in part contingent on there being
potential benefits (for public health or for participants) that outweigh the expected risks. Important
potential benefits to public health include those arising from the acceleration of vaccine development,
the development of more effective vaccines, and the improvement of relevant scientific knowledge that
can inform public health practice. Arguably, the benefit-risk profile of a particular study should also be
considered by regarding its place in an overarching research program, compare favourably with
alternative research designs, and be evaluated in terms of the generalisability of the findings (eg,
estimates of vaccine efficacy) to relevant populations.

The use of human challenge studies in the development of vaccines for SARS is likely to have several
benefits, including the opportunity to directly compare the efficacy of multiple vaccine candidates when
doing multiple field trials might be less efficient or unfeasible. The benefits are likely to be higher when
estimates of vaccine efficacy derived from such studies are generalisable to relevant populations and
there is a clear pathway from human challenge studies to further testing and the timely regulatory
approval, manufacture, and distribution of a novel vaccine. Furthermore, in interepidemic or
interpandemic periods when field trials are unfeasible, human challenge studies might be the only way
to test vaccine efficacy.

2. Potential direct benefit to participants

Although human challenge studies are often characterized as non-therapeutic research in which healthy
volunteers do not directly benefit from study participation, there can sometimes be direct benefits to
study participants. Although, payment of participants might be ethically appropriate, payment is
generally not considered a benefit that would offset risks.

 However, potential direct benefits of being infected with SARS in the course of human challenge studies
would include participants being exposed to less infection-related risk than if they are infected in the
community (eg, because of early diagnosis and medical care) and gaining immunity to future infection in
the context of a high background risk (although more data are needed to clarify the degree and duration
of immunity to SARS).

 Participants might also benefit if they receive an experimental vaccine that turns out to be effective.
Participants' immunity, whether the immunity results from challenge infection or an experimental
vaccine, might also benefit third parties, especially if health-care workers are recruited to participate in
the studies, because this immunity might prevent health-care workers from becoming infected and
subsequently infecting others.
 3. Risks to participants
Participants in human challenge studies might face risks associated with the challenge infection and, in
some cases, the experimental vaccine (or other intervention). Such risks should be minimized eg, via the
restriction of participation in initial studies to healthy young adults and the provision of high-quality
medical care, including intensive care, if required.

 More data are needed for more accurate estimates; however, these values include young adults with
co-morbidities who would be excluded from human challenge studies. There might nevertheless be rare
severe outcomes (eg, potentially fatal respiratory failure requiring ventilation) or lasting harms (eg, long-
term respiratory deficits) among participants in human challenge studies.

 Although these risks might be higher than those associated with many modern human challenge
studies, such risks might be considered acceptable if human challenge studies have considerable
expected benefits, the risks in question do not entail a major net increase in risk (in light of background
risks of infection), and there is long-term follow-up of participants and full compensation for any
research-related harms. SARS challenge studies might thus be ethically acceptable (especially when
participants already face a high background probability of infection), even in the absence of specific or
curative treatment. The use of attenuated strains that could provide data equally as useful as the data
provided by wild-type strains and the use of proven specific treatments (if developed) could further
reduce risks to participants, but developing such strains or treatments could take a long time and thus
detract from the acceleration of vaccine development that is enabled by challenge studies.

4. Self experimentation
It is conceivable that researchers might volunteer for human challenge studies during the current
pandemic alongside other volunteers. This occurrence raises questions regarding the degree to which
self-experimentation increases the permissibility of high-risk human challenge studies. The Nuremberg
Code posits that high-risk studies might be more acceptable when researchers themselves serve as
volunteers. However, this suggestion, which was appealed to in vindication of early yellow fever
studies, might be controversial, in part because clinical and research staff might feel pressure to
participate. Whether the willingness of researchers to undergo the risks of challenge infection would
justify exposing other research participants to higher risks is also unclear.

 In any case, so long as all participants provide adequate informed consent and other research ethics
criteria are met, high-risk human challenge studies might be justified whether or not they include self-
experimenting researchers.

5. Risks related to experimental vaccines

Although vaccines are usually associated with very low risks, experimental vaccines might not protect
participants and, in some cases, might even increase the severity of disease among those who are
subsequently infected. These outcomes have occurred, for example, for vaccines against respiratory
syncytial virus and dengue virus, in some cases resulting in small numbers of deaths among participants
in vaccine research. This kind of danger might apply to corona virus vaccines, because vaccine-enhanced
disease has been observed in animal challenge studies with corona viruses.
Vaccine-enhanced disease could result in high risks to participants in both human challenge studies and
vaccine field trials. Challenge studies are arguably a superior way of evaluating the risk of vaccine-
enhanced disease compared with field trials because smaller numbers of participants are vaccinated and
challenged at a time and participants receive closer monitoring and more immediate medical care than
would be available in a field trial. However, if vaccine-enhanced disease is rare, human challenge studies
enrolling small numbers of participants might not reveal it.
Challenges in vaccine production
1. Animal model
Both large and small animals can be infected with SARS-CoV, a giant advance for vaccine research.
Examining infected animal models will provide information that will lead to an understanding of the
correlates of immunity. Mice have been used to further the understanding of virus neutralization,
cytokine upregulation and the minimum requirements for viral clearance, but have yet to show disease
that mimics the atypical pneumonia seen in adult humans. While there have been some reports of
disease in cynomolgous macaques , many groups have not reproduced these findings. A promising
animal model may be the domestic ferret. Ferrets show elevated liver enzymes, lymphocytic infiltration
and alveolar damage, which has also been observed in humans.

Despite the usefulness of these animal models, many challenges lie ahead. First, animal models of SARS-
CoV infection do not mimic human disease. In mice, the virus is cleared in less than 1 week and minor
pathology in the lung is observed. Histopathology performed on necropsy samples suggests that lung
epithelial cells are involved, although the absence of pneumonia and infiltrating macrophages is
disappointing. Stat1-deficient mice may prove promising as an animal model that most similarly mimics
human disease.

2. Lack of testing population

In order to test efficacy, large human populations must be tested in areas where the virus is endemic.
Finally, if SARS fails to return, how will vaccine manufacturers test candidate vaccines for efficacy? While
the animal rule has been provided for just this type of case, an animal model should mimic human
disease in order to be applicable.

3. Short lived immunity

Corona viruses may induce a short-lived immunity. This may be the reason that humans are subject to
multiple infections with corona viruses that cause the common cold. Long-term immunity studies for
SARS-CoV are currently underway.

4. Antibody Dependent Enhancement (ADE)

Antibody-dependent enhancement (ADE) has been observed in vaccinated and wild-type infections of
FIP. ADE is thought to potentiate viral infection through the infection of macrophages. Viral entry into
macrophages occurs when antibodies bind the virus and attach to macrophages via the Fc region of the
antibody and its interaction with cell surface expressed Fc receptors.

While there has been no observation of ADE during SARS infection, it is worth noting that one
coronavirus, FIPV, is capable of eliciting ADE and in the evaluation of vaccines, we may want to consider
this possible outcome.
 5. Short lived mucosal immune response
Achieving sterilizing immunity at mucosal surfaces is a major challenge to prevent virus shedding and
mucosal immunity is often short-lived, requiring multiple booster vaccine doses. A possible scenario is
that vaccines will prevent severe disease and deaths, but may not eliminate nasal shedding, allowing
continued transmission.

6. Stimulating the right parts of immune system

The human immune system is very delicately balanced. On the one hand it needs to be able to identify
and kill viruses and other infectious organisms. On the other hand it is important that an overactive
immune response does not damage the healthy tissues of the body. Another feature of the immune
system is that it has evolved to be able to fight and protect against a wide variety of infections. For
example, lymphocytes that help in immune responses, called helper T cells, come in several varieties.
The first (called Th1) kills bacteria and viruses that infect cells; the second (Th2) controls larger parasites
like helminths (worms). Activating the wrong pathway can increase the amount of inflammation and
worsen the disease.

As part of normal immune responses, lymphocytes and other cells produce immune signalling chemicals
called cytokines. These co-ordinate and stimulate immune responses. But if these responses are
excessive, they can cause inflammation, which in extreme cases can result in the shut-down of vital
organs including the lungs, heart and kidneys. In the case of the SARS virus, there is evidence that this
may be responsible for some of the late complications of the disease. In a small percentage of those
infected with SARS-CoV-2, an overactive immune response may make an important contribution to the
late complications of COVID-19. However, this is not known yet for certain and is an important area for
research.
Economic analysis
Corona virus family have had major economic impact over the years. A lot of time, effort and money
have been invested in research and development of vaccine for corona virus family including SARS,
MERS and SARS-CoV-2.

Till date no commercial vaccine have been produced for these viruses. But according to Moderna
Pharmaceuticals, a price range of 32$- 37$ per dose have been speculated so far which is very expensive
considering the fact that we still do not know how many booster doses are required to achieve full
immunity. If this price range is true for when the vaccine finally comes out, underdeveloped and
developing countries will not be able to afford it for its entire population.
Conclusion
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