Get best of the week

Wedge by wedge: use of the PIV5 virus to create a vaccine against MERS-CoV and possibly against SARS-CoV-2



Given the current circumstances, it is not at all surprising that the scientific community has concentrated all its forces on the search and development of methods and tools to combat the spreading virus. Today we look at a study in which scientists from the American Society for Microbiology (ASM) describe a very working vaccine against the MERS virus, which began to spread in 2012. According to scientists, their development can help create a vaccine against the currently raging SARS-CoV-2. What is the peculiarity of the created vaccine, from which it was created, and how effectively does it perform its task? We learn about this from the report of scientists. Go.

UFO Care Minute


The pandemic COVID-19, a potentially severe acute respiratory infection caused by the SARS-CoV-2 coronavirus (2019-nCoV), has officially been announced in the world. There is a lot of information on Habré on this topic - always remember that it can be both reliable / useful, and vice versa.

We urge you to be critical of any published information.


Official sources

, .

Wash your hands, take care of your loved ones, stay at home whenever possible and work remotely.

Read publications about: coronavirus | remote work

Study basis


MERS or the Middle East respiratory syndrome is an inflammatory disease of the respiratory organs that develops due to infection with the virus of the genus Betacoronavirus from the now familiar subfamily Coronavirinae.


Snapshot of MERS-CoV (yellow).

The source of the MERS-CoV virus is again bats. The first cases of infection among people were recorded back in 2012 on the territory of the Arabian Peninsula, but by 2015 the virus had reached the countries of East Asia and Europe. According to the latest data, the number of confirmed cases of MERS-CoV infection is 2494 people, among whom 858 died (data from 02.02.2020). The data vary from source to source, but it is clearly seen that the degree of distribution in MERS-CoV is not as great as in SARS-CoV-2. Nevertheless, the Middle East virus also has a rather high mortality rate - about 35%. This indicator is the most worrying.

MERS-CoV is a positive chain RNA virus * , in which penetration into target cells is carried out due to the S-proteinviral envelope * .
Positive chain RNA virus * - positive viral RNA. This implies that a particular viral RNA sequence can be directly translated into viral proteins (e.g., proteins necessary for virus replication). Therefore, in RNA viruses with a positive chain, the viral RNA gene can be considered viral mRNA (messenger RNA) and it can be immediately translated by the host cell.
Viral envelope * - an additional envelope covering the outer envelope (capsid) of many viruses.
The S protein consists of the S1 subunit responsible for binding to the virus receptor, dipeptidyl peptidase 4 (DPP4 or CD26) via the receptor binding domain (RBD from the receptor binding domain ), as well as the S2 subunit, which provides membrane fusion.

Researchers recall that at the moment there is no vaccine against MERS-CoV, there are only a number of developments whose effectiveness is quite doubtful. Scientists believe that their work will change this situation.

In this study, scientists decided to turn their attention to another virus - PIV5. PIV or parainfluenza viruses (PG) have five varieties, three of which cause diseases in humans (from PG-1 to PG-3) and two that are not dangerous for humans (PG-4 and PG-5).


TEM snapshot of parainfluenza virus.

PG-5 is a member of the genus Rubulavirus from the Paramyxoviridae family, which includes mumps virus (MuV or “mumps”) and human parainfluenza virus type 2 (PG-2) and type 4 (PG-4). PG-5 encodes eight known viral proteins. Nucleocapsid protein (NP), phosphoprotein (P) and RNA polymerase protein (L) are important for transcription * and replication * of the viral RNA genome.
Transcription * - the process of RNA synthesis using DNA as a template; transfer of genetic information from DNA to RNA.
Replication * is the process of creating two daughter DNA molecules based on parental DNA.
Scientists believe that PG-5 is an excellent candidate for viral vectors * for the future vaccine. PG-5 is quite safe, despite its high degree of infectiousness, which can play into the hands when creating an effective vaccine.
Viral vectors * are tools for delivering genetic material to target cells.
In addition, PG-5 does not have a DNA phase in its life cycle, which avoids possible inadvertent genetic modifications of the host cell DNA by recombination or insertion. Also, the structure of PG-5 is quite stable, in contrast to RNA viruses with a positive chain. This is confirmed by the created recombinant PG-5 expressing the F gene for the respiratory syncytial virus, when the F gene was maintained for more than 10 generations.

Another advantage of PG-5 is its cost-effectiveness, as this virus can be grown up to 8x10 8 PFU / ml (PFU - plaque forming units).

Previously, PG-5 was already used as a viral vector when working with vaccines against the respiratory syncytial virus and rabies. During experiments in mice, it was found that the PG-5 vector expressing NA (neuraminidase, which is part of the membrane) of the influenza virus, leads to complete immunization of the experimental subjects, i.e. 4 days after infection, there was not a single case of death or even infection. But PG-5, expressing NP (nucleoprotein), protects 100% of experimental mice from the lethal strain of the H1N1 influenza virus.

Based on such positive results from previous studies, scientists decided to use PG-5 as a viral vector expressing the MERS virus S protein. As before, all experiments were carried out on mice.

Research results


Researchers preliminarily introduced the HA (hemagglutinin) gene of the influenza A virus into different places in the PG-5 genome and found that inserts in SH (small hydrophobic) and HN (hemagglutinin-neuraminidase) give a better immune response.
Influenza virus hemagglutinin and neuraminidase * are surface antigens of the virus that provide the ability to attach to target cells.
Given this, it was decided to introduce the full-sized S gene of the MERS virus at the intersection of SH and HN. A plasmid * containing a full-sized cDNA * PG-5 with an inserted S gene at the junction of SH and HN was constructed using standard molecular cloning methods ( 1A ).


Image No. 1
Plasmid * is a small DNA molecule that is separated from chromosomes and capable of independent replication.
cDNA * - complementary DNA, i.e. DNA synthesized on a matrix of mature mRNA (messenger RNA) in a reaction catalyzed by reverse transcriptase.
The plasmid was transfected * into BHK * cells along with plasmids expressing the TG, NP, P and L RNA polymerase of PG-5 virus (PIV5).
Transfection * - the introduction of nucleic acid into cells by a non-viral method.
BHK cells * ( baby hamster kidney ) are connective tissue cells (fibroblast) of hamster kidneys that are sensitive to many viruses that affect humans. BHK cells are used for transformation, as well as for stable and transient transfections.
The result was the infectious virus PIV5-MERS-S, which was further purified and multiplied in large numbers in MDBK cells * for further analysis.
MDBK cells * ( Madin-Darby canine kidney cells ) - dog kidney cells, first isolated in 1958 by scientists SH Madin and NB Darby. These cells are used in research as model mammalian cells.
The virus genome was sequenced and confirmed that it contains the desired input DNA sequence. Next, it was necessary to check the expression of S protein in cells infected with PIV5-MERS-S. For this, the cells were infected at different MOI * values , after which they were lysed (dissolved) for immunoblotting * using an anti-S antibody.
MOI * ( multiplicity of infection ) - the multiplicity of infection, i.e. the ratio of agents (virus) to targets for infection (cells).
Immunoblotting * is a highly sensitive method for detecting proteins, based on a combination of electrophoresis and enzyme immunoassay or radioimmune analysis.
The analysis showed the presence of both full S protein and S2 fragments, which indicates the success of its implementation ( 1B ). S protein expression in PIV5-MERS-S infected cells was further confirmed by immunofluorescence analysis ( 1C ).

Scientists note that PIV5-MERS-S caused massive syncytium * formation in Vero * cells . In addition, the growth kinetics of PIV5-MERS-S and conventional PG-5 (PIV5) was very similar ( 1D ).
Syncytium * is a type of tissue in which cells are not completely separated, i.e. there are sections of cytoplasm with nuclei that are interconnected by plasmodesmata (cytoplasmic bridges).
Vero* — , .


Image No. 2

Immunization PIV5-MERS-S generates neutralizing antibodies and T-cell mediated immunity. To determine whether PIV5-MERS-S can generate immune responses, mice were immunized with a single dose of PIV5-MERS-S or control PIV5-GFP virus at 10 4 PFU or 10 6 PFU per individual via the intranasal pathway.

While both doses elicited antibody responses, neutralizing titers were modest at 1:64 and 1: 128 for 10 4 and 10 6 doses, respectively ( 2A and 2B ).

Scientists recall that mice of the species C57BL / 6 and BALB / c after immunization generate Th1- and Th2-dominant immune responses, respectively.
Th (T-helpers) - T-lymphocytes that enhance the adaptive immune response (acquired immunity).

Th1 - promote the development of a cellular immune response by activating macrophages.

Th2 - activate B-lymphocytes, which helps the development of a humoral immune response.
One dose of PIV5-MERS-S (10 6 PFU) resulted in a titer of neutralizing antibodies up to 1: 2000 in BALB / c ( 2C ) mice , which is consistent with the Th2-dominant response in mice of this species.

To evaluate the primary response of CD8 * T cells generated by immunization with PIV5-MERS-S, hDPP4-KI mice were intranasally immunized with PIV5-MERS-S (10 4 PFU).
CD8* ( 8) — , - .
Four weeks later, a fence was taken to examine the presence / absence of MERS-CoV-specific pulmonary resident CD8 T cells ( 3A ).


Image No. 3

As shown in 3B - 3D , there was a significant increase in the percentage and total number of CD8-IFN cells in the lungs of mice immunized with PIV5-MERS compared to those infected with PIV5-GFP.

To evaluate the response of MERS-S-specific CD8 T cells, an additional MERS-CoV MA injection was performed (10 4 PFU). As a result, a significant increase in the response of CD8 T cells on the fourth day was seen compared to mice infected with the control PIV5-GFP ( 3E - 3G)

A 10-fold increase in MERS-S-specific CD8 T cells was also observed compared to the primary response of CD8 T cells ( 3B - 3G ).


Image No. 4

As a result of PIV5-MERS-S immunization prevents the death of infection in mice. To determine the effectiveness of PIV5-MERS-S in the prevention or modification of the MERS-CoV virus, hDPP4 KI mice were immunized with PIV5-MERS-S (10 4 PFU) by the intranasal route.

4 weeks after immunization, the mice were infected with MERS-S modified to be more active in mice ( 4A ). All mice immunized with PIV5-MERS-S survived this deadly infection, with slight weight loss ( 4B and4C ). In contrast, all mice infected with the control PIV5-GFP or PBS died after infection. This indicates that PIV5-MERS-S fully protected mice from lethal infection.

Despite the fact that PIV5-MERS-S immunized mice had a higher rate of lung clearance from the virus, this method did not provide complete sterilizing immunity ( 4D ).


Image No. 5

Histopathological studies of the lungs after infection showed that mice immunized with PIV5-MERS-S had significantly less cellular debris * and more mononuclear infiltrates ( 5A and 5B ).
Cell debris * is the remainder of the cell surrounded by a plasma membrane that is phagocytosed by macrophages.
Strong leukocyte infiltration (mainly mononuclear cells) and less damage (edema, hyaline membranes, necrotic cell debris, etc.) were also detected.

At the final stage of the study, scientists evaluated the protective responses induced by PIV5-MERS-S or inactivated MERS-CoV. For this, some mice were immunized with PIV5-MERS-S (10 4 PFU) or PBS, while others were immunized with UV-inactivated MERS-CoV.

While PIV5-MERS-S provided 100% protection against lethal infection, inactivated MERS-CoV protected only 25% of mice (image below).


Image No. 6

Next, a study was carried out of the lungs of mice that were first immunized and then infected with MERS-CoV (image below).


Image No. 7

More eosinophils (a type of white blood cell) were observed in the lungs in mice immunized with inactivated MERS-CoV than in mice immunized with PBS or PIV5-MERS-S after infection with MERSCoV.

Compared to PBS, perivascular eosinophilic infiltration was significantly increased in the inactivated MERS-CoV group, but no statistical difference was observed compared to the group immunized with PIV5-MERS-S.

An assessment was also made of the formation of a hyaline membrane in the alveoli of the lungs, which is a clear sign of severe pulmonary disease. Compared to the PBS group, mice immunized with PIV5-MERS-S showed significant protection against hyaline membrane formation. But the mice immunized with inactivated MERS-CoV showed only a slight decrease in the formation of the hyaline membrane, which indicates the low efficiency of this method of immunization, in comparison with PIV5-MERS-S.

For a more detailed acquaintance with the nuances of the study, I recommend a look at the report of scientists .

Epilogue


In this work, scientists were able to demonstrate a quite successful method of immunizing mice with the MERS-CoV virus, which has been walking freely on our planet since 2012.

The main distinguishing feature of the developed method is the use of another virus, namely PG-5, as a viral vector. In other words, the wedge is knocked out by wedge.

The use of the virus as a transport for the delivery of genes necessary for immunization can be considered a rather unusual method, but it can contribute to a more effective fight against viruses. The researchers themselves say that their work is aimed not only at combating MERS-CoV, but also with SARS-CoV-2 (COVID-19). Both viruses belong to the same subfamily, have similar infection pathways and effects on target cells. If the developed method of immunization is further developed, then it may be possible to create a sufficiently effective vaccine.

This is what the authors of this work are planning to do in the future. Whether they succeed in creating a super vaccine or not, time will tell. However, now they are worthy of all praise at least for paying attention to the virus, about which everyone seemed to forget.

Be that as it may, we can only hope that science and everyone involved, like many times before, will be able to overcome all prejudices and fears, overcome all obstacles and show that analytical thinking, based on knowledge and logic, is always capable of find a way out of any current situation, no matter how terrible at first glance it may be.

Thank you for your attention, remain curious and have a good working week, guys.

A bit of advertising :)


Thank you for staying with us. Do you like our articles? Want to see more interesting materials? Support us by placing an order or recommending to your friends cloud-based VPS for developers from $ 4.99 , a unique analog of entry-level servers that was invented by us for you: The whole truth about VPS (KVM) E5-2697 v3 (6 Cores) 10GB DDR4 480GB SSD 1Gbps from $ 19 or how to divide the server? (options are available with RAID1 and RAID10, up to 24 cores and up to 40GB DDR4).

Dell R730xd 2 times cheaper at the Equinix Tier IV data center in Amsterdam? Only we have 2 x Intel TetraDeca-Core Xeon 2x E5-2697v3 2.6GHz 14C 64GB DDR4 4x960GB SSD 1Gbps 100 TV from $ 199 in the Netherlands!Dell R420 - 2x E5-2430 2.2Ghz 6C 128GB DDR3 2x960GB SSD 1Gbps 100TB - from $ 99! Read about How to Build Infrastructure Bldg. class c using Dell R730xd E5-2650 v4 servers costing 9,000 euros for a penny?

More articles:


All Articles