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Characterization of markers of cold-adapted candidate virus strains for live attenuated vaccines against chickenpox and shingles

https://doi.org/10.36233/0372-9311-2020-97-4-2

Abstract

Introduction. Chickenpox poses a significant public health concern due to its worldwide occurrence, a substantial probability of severe clinical progression, development of complications that can lead to a fatal outcome. Routine vaccination is the only way to prevent the disease. The purpose of this study was to assess the attenuation of cold-adapted (CA) candidate virus strains of Varicella zoster and Herpes zoster by using traditional and new methods.
Materials and methods. The study was performed on strains of diploid cells from human embryonic lung and musculocutaneous tissue, primary and diploid cells of guinea pig fetal fibroblasts. Two clinical isolates of the virus were obtained — from a child with chickenpox and from an adult during the reactivation of shingles. The vOka vaccine strain and Ellen strain, a laboratory strain, were used as a control. The viral infectivity was measured by using a sensitive limiting dilution assay. The virulence was measured through the analysis of chick embryo chorioallantoic membranes infected with the Varicella zoster virus.
Results. The clinical isolates were sub-cultured at lower temperatures, put through comparative tests and checked for presence of attenuation biomarkers. It was found that vFiraVax, a Varicella zoster virus strain, and vZelVax, a Herpes zoster virus strain were temperature-sensitive and cold-adaptable, but they lacked virulence. Attenuated CA virus strains induced lower expression of IFN-α and IFN-γ receptors on human mononuclear cells as compared to their parental variants.
Conclusion. We created and assessed two candidate vaccine strains through attenuation of clinical isolates.

Introduction

The Varicella zoster virus (VZV) is a human α-herpesvirns and is closely related to herpes simplex virus types 1 and 2. VZV has a typical morphology of the herpes virus, but its genome is the smallest one among α-herpesviruses and it encodes more than 70 gene products [1].

Chickenpox (CP) has common etiology and a close pathogenic relationship with the chronic form of the infection — shingles, also known as herpes zoster (HZ), caused by the Herpes zoster virus (HZV). CP and HZ are different clinical forms of the same infection process. The risk of HZ development in those who had CP in the past is 10-30%. Among people aged 60-80 years, the HZ incidence ranges from 5 to 10 cases per 1,000 people of the above age. 15-40% of HZ patients develop postherpetic neuralgia, which is very difficult to treat and which can substantially affect quality of life. Other complications caused by the reactivation in­clude encephalitis, motor weakness, myelopathy, etc. The most severe complications are observed in immu­nocompromised patients [2][3][4].

For many years, foreign healthcare has been us­ing live culture-derived vaccine based on the attenua­ted vOka virus strain (Japan). This vaccine helps build up long-term high-level post-vaccinal immunity after the two-time immunization cycle (the follow-up peri­od during 10-20 years) and prevents fatal outcomes in newborns and children with a poor immune system; it also partially prevents development of HZ in elderly people and people with low immunity [5].

The vaccine for immunization of children and adults is based on the vOka vaccine strain. The VZV concentration in the vaccine for adults is increased 2 times.

There are no comparable home-made CP vaccines in Russia.

At our laboratory, we created two attenuated VZV vaccine strains for development of live culture-derived vaccines for children and adults.

The main purpose of this study is assessment of attenuated vaccine strains by using traditional biomark­ers of attenuation, while looking for new attenuation markers for more comprehensive monitoring of prop­erties inherent in candidate vaccine viral strains for the live culture-derived vaccine against CP and HZ.

Materials and methods

Cell cultures. The study was performed on strains of diploid cells from human embryonic lung (HEL-3 and MRC-5) tissue, strains of diploid cells from human embryonic musculocutaneous tissue (MC 27), prima­ry and diploid cells from guinea pig fetal fibroblasts (GPFF).

Diploid cell cultures were grown in DMEM/F12 medium (PanEco) with 10 mM HEPES, 5% fetal calf serum (HyClone) with addition of 2 mM glutamine and 40 μg/ml gentamicin.

Viruses. The clinical VZV isolate and clinical HZV isolate were obtained from infected patients in the form of vesicle scabs formed after the appearance of the rash. One isolate was obtained from a healthy 6-year-old girl who became ill with CP in Moscow; the other isolate was obtained from an adult 63-year-old man during the reactivation of recurrent HZ.

Attenuation of clinical isolates. The virus iso­lates of CP and HZ were attenuated on HEL-3 cells at a low temperature (30°C). The isolates underwent 12 passages on HEL-3 diploid cells, 6 passages on prima­ry GPFF cells and, additionally, 2 passages on HEL-3 cells. After the completion of the full cycle of cold ad­aptation, the VZV isolate was named FiraVax VZV and the HZV was named vZelVax HZV.

The attenuated vOKA/Merck VZV vaccine virus strain (USA) and the laboratory Ellen VZV virus strain (USA) were used as VZV reference-viruses.

Estimation of VZV infectivity. The viral infectivity was estimated on MC-27 or GPFF cell cultures grown on 24-well plates. The DMEM maintenance medium with 2% fetal calf serum (FCS) was used to prepare tenfold dilutions of virus-containing fluid (VCF) from 10–1 to 10–10. The medium was removed from the plates with grown cells, and the cell monolayer was flushed one time with phosphate-buffered saline (PBS). Then, 0.1 ml of each VCF dilution was added to the center of the well with cell monolayer and left for contact at 36.5°C and 5% CO2 for 1 hour. Upon completion of the contact, 1 ml of DMEM maintenance medium with 2% FCS was added to each well, including wells with con­trol cells. The titration results were measured on the 6th-7th day from the time of infection by using a hemad­sorption test performed with 0.25% suspension of red blood cells from guinea pig or human type 0 positive blood (Rh+). The virus titer corresponded to the highest virus dilution causing hemadsorption in 50% of infec­ted cultures, with no hemadsorption in control uninfec­ted cell cultures.

Infection of the chick embryo chorioallantoic membrane (CAM). An artificial air sac was made in 11-12-day-old chick embryos to apply 0.1 ml of VZV VCF. The embryos were checked daily for their via­bility. If any embryos died on the next day, they were disposed. The embryos were monitored for maximum 6 days. After infected embryos had been cooled down, their CAMs were removed and placed in a petri dish; the membranes were flushed with PBS and examined for presence of hemorrhage or white pockmarks.

Obtaining VZV-specific mouse sera. Specific pathogen-free BALB/c mice (SPF-mice) were immu­nized intraperitoneally with 0.5 ml of VCF at a con­centration of 6 lg HAU50/0,1 ml (HAU is a hemadsorbing unit) three times a day for 3 days. This immunization cycle was repeated three times at a 2-week interval.

Identification of virus-specific antibodies in an indirect enzyme-linked immunosorbent assay. Lysates of MC-27 cells infected with a laboratory Ellen VZV strain were adsorbed on the solid phase of the 96-well immuno plate as a VZV-sorbent. Lysates of infected cells were obtained through infecting the monolayer of MC-27 cells grown in culture flasks with a surface area of 175 cm2 (Costar). To get the cell monolayer infected. the medium was removed from the culture flasks; then, the cell monolayer was flushed twice with PBS and 5 ml of Ellen VZV VCF were applied to it at a multi­plicity of infection of 0.2. The infected cells were in­cubated for 10 days at 36.5°C and 5% CO2 Then, VCF was collected from the culture flasks; 5 ml of PBS was added to the culture flasks with infected cells. The cul­ture flasks with infected cells were frozen down three times at 70°C and thawed at 4°C. The resulting infected lysate containing cell-associated VZV was examined with NanoPhotometer NP80-Touch spectrometer to measure the protein concentration.

After being vortexed, the infected cell lysates were adsorbed onto the wells of 96-well plates, 50 μl per well at 5 μg protein concentration and dried in a thermostatic oven at 36.5°C for 18-20 hours.

The indirect enzyme-linked immunosorbent assay was performed following standard procedure: Blocking non-specific binding with 1% bovine serum albumin and 0.01 M PBS in a thermostatic oven for 1.5 hour; removal of the blocking solution; titration of the ex­amined guinea pig serum in two steps in the long row of a 96-well plate, from 1: 100 to 1: 204 800 dilution. The process of binding between solid-phase antigens and immune sera took place in the thermostatic ov­en and continued for 1.5 hour. After the contact was completed, the flasks were flushed three times with 0.01 M PBS and 0.05% Tween-20, 200 μl per well. Im­mune complexes were identified with the help of the Protein A-Peroxidase conjugate (Sigma), 100 μl of 1: 500 dilution per well; time in a thermostatic oven 1 hour. Then, the plates were flushed five times with 0.01 M PBS and 0.05% Tween-20 to detect immune complexes by using tetramethylbenzidine substrate solution and hydrogen peroxide for 15 min in the dark. 4N H2SO4 was used to discontinue the reaction. The op­tical density was measured with a Bio-Rad 680 micro­plate reader at the 450-nm wavelength.

Estimation of receptor expression for human α and γ interferons (IFN). The process of isolation of lymphocytes from human venous blood, type 0 positive blood (Rh+), and preparation of samples for a direct immunofluorescence assay were described in detail in the following studies [6][7]. Human mononuclear cells (HMC) were isolated from heparinized blood by using Ficoll gradient centrifugation at a density of 1.077 g/cm3 (PanEco). The obtained HMC were resuspended in RP- MI-1640 medium with 1% bovine serum albumin; then they were induced with VZV antigens and incubated at 36.5°C and 5% CO2. Samples induced with HMC VZV were prepared for a direct immunofluorescence assay at different time intervals. The prepared samples were stained with FITC conjugates based on monoclonal anti-idiotypic antibodies structurally imitating human IFN-α/β and IFN-γ. These anti-idiotypic antibodies are anti-IFN-α/β and IFN-γ antibodies. After being la­beled with labeling reagents, they were examined with an Optika fluorescence microscope at a wavelength of 510-550 nm.

Obtaining VZV hemagglutinin by using E. Nor- rby’s method [8]. Hemagglutinin was received from Ellen VZV VCF to perform a hemagglutination inhibi­tion (HAI) assay for specific anti-VZV-immune sera [8].

VCF was obtained through infecting Vero-CCL-81 cell cultures with laboratory Ellen VZV strains. VCF was clarified by centrifugation at 1,500 rpm for 30 min and treated with Tween-80 and ether. The treated flu­id was centrifuged one more time at the same param­eters to result in forming separate layers. The hemag- glutinating antigen formed the lower layer, which was carefully pumped out into a flask, which was further covered with sterile gauze pad and left for the night to get free from ether vapor. The hemagglutination assay (HA) with suspension of red blood cells from guinea pigs was performed for detection of the titer of virus hemagglutinin.

The HA assay was used to select the required di­lution of the hemagglutinating antigen for the further HAI assay. The latter was aimed to delay the hemagglutinating effect of the virus antigen by specific immune sera.

The HAI assay. The assay was performed with the antigen dilution containing 2 units of antigen in 0.25 ml. Prior to the assay, the studied sera were cleared from heat-labile (by heating at 56°C for 30 min) and heat-re­sistant (by treatment with cholera vibrio filtrate) inhi­bitors. The assay was performed in accordance with the standard procedure. The highest serum dilution sup­pressing the hemagglutinating activity of the antigen was taken as a titer.

The immunogenicity of cold-adapted (CA) vFiraVax VZV and vZelVax HZV vaccine strains was assessed by using an animal model. One dose of the vaccine against CP and HZ was injected subcutaneously into guinea pigs weighing 300-400 g. 2, 3 and 5 months after the immu­nization, blood samples were taken from their hearts to be used in the neutralization test and HAI assay.

The heat-labile and heat-resistant inhibitors were removed from the immune sera for the further neutral­ization test and HAI assay. The VariVax vaccine (USA) containing 1,000 PFU50/0,5 ml or 6 lg HAU50/0,5 ml was used as a neutralizing virus in the neutralization test.

The neutralization test was performed on MC-27 cells. The neutralization test results were recorded on the 7th day after the beginning of the test; the titer was based on 100% protection of cell cultures.

The statistical processing was performed by using Student’s t-test.

Results

During their cold adaptation, viruses normally develop mutations in terms of temperature-sensitivity (ts-phenotype) and cold-adaptation (ca-phenotype). These two phenotypes are the main laboratory determi­nants for candidate vaccine strains for live virus vac­cines or, in other words, the main biological markers of attenuated virus strains [9].

Tables 1 and 2 show the results of titration of at­tenuated and wild-type parental VZV and HZV strains on diploid GPFF and MRC-5 cells.

 

Table 1. Identification of ts- and ca-markers of biological attenuation of candidate VZV vaccine strains on GPFF cells

Viral strain The VZV and HZV infectious titer at different temperatures, lg HAU50/0,1 ml
30°С

36°С

39°С

vFiraVax VZV, 19th passage, vaccinated liquid, 11 days

9,0

7,75

0

vFiraVax VZV, 19th passage, infected cells, 11 days

12,8

11,8

0

pFiraVax VZV, 2nd passage, vaccinated liquid, 11 days

7,75

7,5

6,5

vZelVax HZV, 19th passage, vaccinated liquid, 11 days

8,5

6,5

0

vZelVax HZV, 19th passage, infected cells, 11 days

12,8

10,3

0

pZelVax HZV, 2nd passage, vaccinated liquid, 11 days

7,5

7,5

6,5

vОКА/Merck (USA)

6,5

5,5

0

Note. Here and in Tables 2-6: v — a vaccine strain, p — a parental strain.

 

 

Table 2. Identification of ts- and ca-markers of biological attenuation of candidate VZV vaccine strains on MRC-5 cells

Viral strain

The VZV and HZV infectious titer at different temperatures, lg HAU50/0,1 ml

30°С

36°С

39°С

vFiraVax VZV, 19th passage, vaccinated liquid, 11 days

9,5

7,75

0

vFiraVax VZV, 19th passage, infected cells, 11 days

12,8

10,8

0

pFira VZV, 2nd passage, vaccinated liquid, 11 days

8,0

7,25

6,5

vZelVax HZV, 19th passage, vaccinated liquid, 11 days

8,5

7,5

0

vZelVax HZV, 19th passage, infected cells, 11 days

12,3

11,3

0

pZel HZV, 2nd passage, vaccinated liquid, 11 days

8,0

8,0

6,5

vOKA/Merck (USA)

6,5

6,0

0

The results given in Tables 1 and 2 demonstrate that candidate vFiraVax VZV and vZelVax HZV vac­cine virus strains were not reproduced at the non-permissive temperature of 39°C, i.e. they were tempera­ture-sensitive — ts-phenotype, and were reproduced more efficiently at the sub-optimal temperature, i.e. they were cold-adapted — ca-phenotype.

Candidate vaccine strains should also differ in their ability to be reproduced in the GPFF cell culture.

Attenuated VZV strains must have higher reproductive activity in these cells as compared to wild-type virus isolates.

Table 3 shows the results for identified titers of attenuated and parental VZV and HZV viruses that are reproduced in GPFF cells and are assessed by the he­madsorption test on diploid GPFF cells.

 

Table 3. Comparative reproductive activity of attenuated and parental VZV virus strains in GPFF cells

Viral strain

Infectious titer VZV
and НZV, lg HAU50/0,1 ml

vFiraVax VZV, 19th passage, vaccinated liquid, 14 days

8,5

pFira VZV, 2nd passage, vaccinated liquid, 14 days

6,5

vZelVax HZV, 19th passage, vaccinated liquid, 14 days

8,0

pZel HZV, 2nd passage, vaccinated liquid, 14 days

6,5

The results given in Table 3 show that the infectivity of parental VZV variants, which was estimated on GPFF cells, is by 1.5-2.0 lg HAU50/0,1 ml lower than the infectivity of the attenuated VZV strains. This parame­ter pointed at another biomarker of attenuation of can­didate vaccine strains for live culture-derived vaccines.

We have developed new markers of biological at­tenuation of VZV candidate vaccine strains.

Monoclonal anti-idiotypic antibodies targeting IFN-α/β and IFN-γ receptors on human immune-com­petent cells, which were induced in vitro by VZV, were used for assessment of quantitative indicators of the ex­pression of IFN-receptors [6][7]. Earlier, we used the model of different measles virus strains to show that the level and duration of expression of IFN-receptors were inversely related to the attenuation level of measles vi­ruses [10].

The study included the comparative assessment of the expression of IFN-α/β and IFN-γ receptors on pe­ripheral blood HMC induced by candidate CA-strains of vFiraVax VZV and vZelVax HZV at early and late passage levels. The results of the study are given in Ta­bles 4 and 5. The obtained data showed that both CA- strains of the VZV virus induced a lower expression of IFN-α/β and IFN-γ receptors as compared to their pa­rental variants. This biological marker of attenuation of VZV candidate strains was named an express-IFN-phenotype. This phenotype can also be used for other atte­nuated virus vaccine strains.

 

Table 4. Comparative expression of IFN-α/β receptors (%) on HMC induced in vitro by attenuated and parental VZV strains at different passage levels

Viral strain

Expression of IFN-α/β receptors on HMC at different time intervals (hours)

3

24

48

72

pFira VZV, 3rd passage

14,8 ± 0,9

23,9 ± 1,3

20,0 ± 0,6

6,6 ± 0,7

vFiraVax VZV, 17th passage

3,6 ± 0,5*

8,1 ± 0,4**

4,5 ± 0,4**

1,6 ± 0,3**

pZel HZV, 3rd passage

13,5 ± 0,9

13,4 ± 1,0

17,8 ± 0,8

6,4 ± 0,5

vZelVax HZV, 17th passage

4,3 ± 0,7**

9,4 ± 1,0*

9,0 ± 0,3**

4,3 ± 0,7*

Ellen# VZV

27,8 ± 3,2

18,8 ± 1,4

15,8 ± 2,3

11,6 ± 1,5

Note. Here and in Table 5: The expression of IFN-receptors was estimated through calculating the percentage of light-producing cells in the total number of 2,000 cells, using 4 HMC samples for each time interval. # The virus was concentrated by ultracentrifugation and purified in sucrose density gradient.

* p < 0.05, **p < 0.001 as compared to the parental strains.

 

 

Table 5. Comparative expression of IFN-Yreceptors (%) on HMC induced in vitro by attenuated and parental VZV strains at different passage levels

Viral strain

Expression of IFN-γ receptors on HMC at different time intervals (hours)

3

24

48

72

pFira VZV, 3rd passage

5,8 ± 1,0

14,9 ± 1,1

15,1 ± 0,9

12,0 ± 0,4

vFiraVax VZV, 17th passage

3,6 ± 0,5*

8,1 ± 0,4**

4,5 ± 0,4**

6,0 ± 0,3**

pZel HZV, 3rd passage

7,9 ± 0,8

14,4 ± 0,9

14,3 ± 1,2

11,13 ± 0,5

vZelVax HZV, 17th passage

4,8 ± 0,9*

4,9 ± 0,9**

7,1 ± 0,9**

5,2 ± 0,02**

Ellen VZV

16,6 ± 1,6

12,5 ± 0,35

10,4 ± 0,6

9,0 ± 0,71

Another biological marker of attenuation for VZV vaccine strains is assessment of virulence of attenuated strains (att-phenotype). We have found that when chick embryo CAM VCF was infected by a wild-type virus, it caused death of the embryos; if the embryos stayed alive, CAM developed extensive hemorrhage of blood vessels. When CAM VCF was infected by attenuated VZV strains, CAM displayed white pockmarks.

Immunogenicity is an important feature of attenua­ted vaccine virus strains. Table 6 shows the results of immune serum titers in the neutralization test and HAI assay.

 

Table 6. Immunogenicity of attenuated vFiraVax VZV and vZelVax HZV vaccine strains through immunization of guinea pigs and a neutralization test on cell culture and in HAI assay at different time intervals

Immune sera for VZV vaccine strains

Neutralization test (at 100% protection)

HAI assay (hemagglutinin, Ellen VZV strain)

time since immunization, months

2

3

5

2

3

5

vFiraVax VZV, 20th passage

1 :1600

1 :1600

1 :1600

1 :25600

1 :12800

1 : 6400

vZelVax HZV, 20th passage

1 :1600

1 :1600

1 :1600

1 :25600

1 :12800

1 :6400

Neutralizing VZV strain vOka, 1000 doses

6.0 lg HAU50/0,1 ml

The results given in Table 6 show that the neu­tralizing activity of immune sera specific to candidate vFiraVax VZV and vZelVax HZV vaccine strains was high both in the neutralization test and in the HAI assay. It should be noted that titers of immune sera in the neu­tralization test remained high during the 5 months of the study, while the titers of immune sera in the HAI as­say demonstrated a 4-fold drop by the end of the study. Virus hemagglutinins are known to be less stable than virus-neutralizing antibodies.

Discussion

We studied biological markers of attenuation of the new CA-candidate VZV and HZV strains for developing live culture-derived vaccines to prevent CP among chil­dren and HZ among adults of over 50 years of age.

Tests proved that both CA-strains had main bio­logical markers of attenuation: ts- and ca-phenotype (Tables 1, 2). VZV virus strains have one more phe­notype, which we named cell-phenotype, meaning changes in tissue tropism. Wild-type variants of clin­ical VZV isolates are generally characterized by low­er reproductive activity in primary cells of guinea pig embryos as compared to attenuated virus strains. Tests demonstrated that CA-strains of vFiraVax VZV and vZelVax HZV had a cell-phenotype (Table 3).

At present, there is no model for assessment of VZV virulence — att-phenotype. We have found that chick embryo CAM can serve as a model. It was found that parental variants of VZV viruses cause death of embryos or extensive hemorrhage in CAM, while CA-virus strains of VZV caused formation of white pockmarks. Candidate CA-strains of vFiraVax VZV and vZelVax HZV demonstrated their non-virulence and had an att-phenotype. This technique can be used for assessment of genetic stability of VZV vaccine vi­rus strains.

We have offered a new marker of biological atten­uation of candidate vaccine strains. Earlier, we devel­oped a new methodological approach to assessment of the functional status of the IFN system [6][7]. We used highly sensitive and specific fluorescent probes based on monoclonal anti-idiotypic antibodies imitating bio­logical effects of human IFN-α/β and IFN-γ. The ex­amination of blood samples from donors with different blood types did not find any expression of IFN-α and IFN-γ receptors on HMC, thus suggesting the balanced functioning of the IFN system [7]. When HMC of do­nor peripheral blood were induced in vitro by different measles virus strains, it was found that the level and du­ration of expression of receptors for different IFN types were inversely related to the attenuation of the measles virus [10].

In our tests, CA-strains of VZV and HZV ex­pressed a lower level of IFN-α/β and IFN-γ receptors on HMC membranes (Table 4 and 5); these results were consistent and stable, letting us conclude that the above express-interferon-phenotype is typical of all attenuat­ed vaccine strains.

The most important property of live CA-vaccines is their effectiveness that exceeds that one demonstrat­ed by inactivated vaccines, as they are able to produce more effective innate and adaptive humoral and cellular immune responses [11][12].

The immunogenicity of attenuated vFiraVax and vZelVax vaccine strains was studied through subcu­taneous immunization of guinea pigs. The humoral immune response was assessed in neutralization tests against 1,000 virus doses present in the OkaVax/Merck vaccine (USA) and in the HAI assay against 2-hemag-glutinating units of hemagglutinin received from the laboratory Ellen VZV virus strain. CA-vaccine strains of vFiraVax VZV and vZelVax HZV demonstrated high immunogenicity during 5 months of monitoring (Table 6).

Thus, we have created and assessed two ca-vaccine strains: vFiraVax VZV and vZelVax HZV — can­didates for creating live culture-derived vaccines to prevent CP among children and HZ among adults.

References

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About the Authors

Firaya G. Nagieva
I. Mechnikov Research Institute for Vaccines and Sera
Russian Federation
D. Sci. (Med.), Assoc. Prof., Head, Laboratory of hybrid cell cultures, Department of virology


Elena P. Barkova
I. Mechnikov Research Institute for Vaccines and Sera
Russian Federation
PhD (Biol.), leading researcher, Laboratory of hybrid cell cultures, Department of virology


Alexandra D. Stroeva
I. Mechnikov Research Institute for Vaccines and Sera
Russian Federation
junior researcher, Laboratory of hybrid cell cultures, Department of virology


Alexander V. Sidorov
I. Mechnikov Research Institute for Vaccines and Sera
Russian Federation
PhD (Biol.), Head, Laboratory of genetics of DNA-containing viruses, Department of virology


Vitaly V. Zverev
I. Mechnikov Research Institute for Vaccines and Sera
Russian Federation
D. Sci. (Biol.), Prof., Academician of the Russian Academy of Sciences, Academic Adviser


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