CN118147089B - Attenuated vaccine strain of Katta virus and application thereof - Google Patents

Abstract

The invention discloses a attenuated vaccine strain of a Kappaphycus alvarezii and application thereof. The vaccine strain is a virus with partial base deletion in a nsP3 region, which is obtained by carrying out low-temperature passage on Vero cells and multiple rounds of plaque screening on the basis of a high-virulence Gata virus HN strain, and the plaque of the virus on the Vero cells is obviously reduced, and the attenuated vaccine strain is named GETV-LA11. The attenuated vaccine strain of the present invention may be used in preparing live vaccine, inactivated vaccine, etc. The attenuated vaccine strain GETV-LA11 of the invention has the advantages of stable heredity, good safety, high protection efficiency, and the like, and is beneficial to differential diagnosis; meanwhile, a single dose of attenuated virus can provide complete virus attack protection for newborn piglets through passive immunization of sows, and the attenuated virus strain is a very excellent candidate virus strain for preparing attenuated live vaccine of the Kataa virus.

Description

Attenuated vaccine strain of Katta virus and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a attenuated vaccine strain of a Kappaphycus alvarezii and application thereof.

Background

The cover tower virus (Getah Virus, GETV) has been widely used in the breeding of animals in recent years, resulting in a great economic loss to the pig industry. In addition, GETV was detected in cattle, horses, pandas, blue fox, or other arthropods, and GETV neutralizing antibodies were also detected in humans. Therefore GETV is a mosquito-borne zoonotic virus with a wide host range, threatens the healthy development of the breeding industry and has potential zoonotic potential.

At present, the prevention and control measures for the Katavirus disease are imperfect, and GETV infection is easy to ignore in screening of animal clinical diseases (such as abortion and diarrhea). In addition, there is currently no approved commercial GETV vaccine for the prevention and control of porcine circovirus disease, and there is a need to develop an ideal vaccine for GETV prevention and control. Live attenuated vaccines generally have the advantage of single dose, high immunogenicity, but careful consideration of their safety is required, especially in attenuated strains that rely on small numbers of point mutations to more readily revert to virulence phenotype.

In summary, according to the clinical practical needs, in combination with the current situation that no GETV vaccine is available and the potential interspecies transmission risk of GETV, there is an urgent need to develop a new safe and effective GETV vaccine to cope with the widely popular GETV wild strain.

Disclosure of Invention

One of the purposes of the invention is to identify a strain of attenuated strain of the katavirus (Getah Virus) GETV-LA11 with partial base deletion of nsP3 by means of Vero cells low-temperature serial passage combined with multiple rounds of plaque screening;

the second object of the invention is to provide the application of the attenuated vaccine strain of the cover tower virus.

It is still another object of the present invention to provide a vaccine comprising the attenuated strain of the foregoing katavirus.

It is a fourth object of the present invention to provide the use of the vaccine described above.

The above objects of the present invention are achieved by the following technical solutions:

in a first aspect, the present invention utilizes a GETV virulent strain isolated from the body of a diseased pig (GenBank accession number: MZ736801 of NCBI), first serially passaged at low temperature on Vero cells, passed to 90 generations followed by a round of plaque screening every 10 generations, and finally obtaining a GETV strain at 130 th generation, in which the base is deleted in the nsP3 gene, which is designated as Gattavirus (Getah Virus) GETV-LA11.

The previously mentioned Katta virus (Getah Virus) GETV-LA11 is now deposited in China center for type culture Collection with the following deposit address: chinese, wuhan, university of Wuhan, accession number: cctccc NO: v202442, the preservation time is 2024, 4, 9 days.

In a second aspect, the invention provides the use of a attenuated strain of katavirus as hereinbefore described for the preparation of a katavirus vaccine.

In specific embodiments, the vaccine is a live attenuated or inactivated vaccine against the cover virus.

In a specific embodiment, the vaccine comprises the attenuated strain of the katavirus as described above as the only active ingredient or one of the active ingredients.

In a third aspect, the invention also provides the use of a attenuated strain of the virus of the genus kataa as described hereinbefore for the manufacture of a medicament or agent for the treatment, prophylaxis or diagnosis of a disease caused by kataa virus.

In a fourth aspect, the invention provides a vaccine comprising a attenuated strain of katavirus as hereinbefore described.

In a specific embodiment, the vaccine further comprises pharmaceutically acceptable excipients.

In a fifth aspect, the invention also provides the use of a vaccine as hereinbefore described for the manufacture of a medicament or agent for the treatment, prophylaxis or diagnosis of a disease caused by a katavirus.

Advantageous effects

(1) The invention screens out a strain of attenuated vaccine with the gene deletion of the Katta virus through low-temperature continuous passage and multiple rounds of plaque, 108 bp deletion exists in the nsP3 gene, the specific position in the genome is 5326 nt-5433 nt, and the genome sequence GenBank accession number of the parental virus GETV-HN strain is MZ736801; deletion of large gene segments greatly stabilizes the genome of the kappaphycus alvarezii, making it less susceptible to reversion to virulence phenotype.

(2) The safety of the Katavirus GETV-LA11 of the present invention was comprehensively evaluated using a GETV sensitive neonatal suckling mouse model and a piglet model. The results indicate that high doses of GETV-LA11 are not lethal in neonatal rats and do not cause weight loss. GETV-LA11 showed strong immunogenicity in a 3-week-old mouse model and had complete protection against subsequent challenge with the high-dose parent GETV-HN strain.

(3) GETV-LA11 also has obviously reduced toxicity in piglets, does not cause clinical symptoms such as diarrhea and the like, and shows good serum conversion 14 days after toxicity attack. In addition, GETV-LA11 strain is safe in pregnant sows, does not cause side effects such as abortion and the like, but maintains good immunogenicity, so that the new generation offspring of the strain is passively protected from infection of the high-dose parent GETV-HN strain by sucking breast milk. The result shows that the gene deletion virus (GETV-LA 11) obtained in the invention is a attenuated strain with good safety and immune protection efficacy, and can be used as attenuated vaccine.

Preservation information

Strain name: the Kappaphycus alvarezii (Getah Virus) GETV-LA11;

Preservation mechanism: china center for type culture Collection;

The preservation organization is abbreviated as: cctccc;

preservation address: chinese university of Wuhan;

Preservation date: 2024, 4 and 9;

accession numbers of the preservation center: cctccc NO: v202442.

Drawings

FIG. 1 shows the results of PCR identification of the C fragment of GETV-LA 11.

FIG. 2 shows the results of IFA identification GETV-LA 11.

FIG. 3 shows the results of plaque assay GETV-LA 11.

FIG. 4 is a multi-step growth curve of GETV-LA11 on BHK-21 cells.

FIG. 5 is a pathogenicity experiment of GETV-LA11 in neonatal ICR rats; wherein, the A graph in FIG. 5 shows the survival rate, and the B graph in FIG. 5 shows the weight change.

Figure 6 is IgG antibody detection in surviving piglets.

FIG. 7 is an immunoprotection experiment of GETV-LA11 in 3-week-old ICR mice. Wherein, panel a in fig. 7 is IgG antibody detection, panel B in fig. 7 is viremia after reinfection, and panel C in fig. 7 is spleen viral load 3 days after reinfection.

FIG. 8 is an antibody assay for GETV-LA11 immunized pregnant sows.

Detailed Description

The present invention will be described in further detail with reference to examples. The reagents or instrumentation used are not manufacturer specific and are considered to be commercially available conventional products.

Example 1 acquisition of the strain LA11 of the Katavirus and molecular characterization

Inoculating single-layer Vero cells with good growth of P1 generation after 3 times of purification of the Gata virus HN strain (GenBank accession number: MZ736801 of NCBI), placing in a CO ₂ cell incubator at 28 ℃, sterilizing after 80% of cells have lesions, freezing the culture dish to a refrigerator at-80 ℃, centrifuging at 13000 Xg for 5 min to remove cell fragments after freezing and thawing, and continuing to passage. When the virus gradually adapts to low-temperature growth, 36-48 h can harvest the virus.

When the virus was passaged to 90 passages, smaller diameter plaque phenotype screening was performed every 10 passages. The specific method comprises the following steps: diluting the harvested virus liquid, inoculating the diluted virus liquid into a 6-hole plate Vero after diluting according to a ratio of 1:10 ⁵, discarding the virus liquid after incubating for 1 h at 37 ℃, replacing the DMEM culture liquid containing 1% of low-melting agarose, placing the DMEM culture liquid into an incubator for continuous culture, picking up 3 plaques with the smallest single diameter for virus propagation when obvious plaques appear in 72-84 h, extracting a virus genome after the propagation, amplifying by using primers for amplifying the whole genome, amplifying by using the extracted single plaque virus genome as a template, and observing the size of a strip or carrying out measurement and identification. Viruses containing more mutations or more amino acid deletions were used for continued passage. By analogy, when the low-temperature passage is combined with plaque screening to 130 generations, a significantly reduced band is detected by PCR (figure 1), and virus liquid amplified by the clone is stored as a new P0 virus and is named GETV-LA11 strain. Sequencing results show that 108 bp of the virus is deleted at the C end of nsP3, and the toxicity of different alphaviruses is influenced by taking account of heterogeneity of the C end of nsP3 protein, so that GETV-LA11 strain has potential toxicity attenuation possibility, and GETV-LA11 strain is inoculated to BHK-21 cells for later use after propagation.

GETV-LA11 was deposited with the China center for type culture Collection with the following deposit address: chinese, wuhan, university of Wuhan, accession number: cctccc NO: v202442, the preservation time is 2024, 4, 9 days.

Example 2 IFA identification of the strain LA11 of the Katavirus

The GETV-LA11 strain with moi=0.01 was inoculated into BHK-21 cells in 96-well plates and IFA identification was performed using GETV E2-3H2 mab after infection with 12H: the infected cells were fixed 15min with 4% paraformaldehyde, then permeabilized 10 min with 0.1% Triton X-100, washed 3 times with PBST; then adding 5% skimmed milk powder for room temperature sealing 1h, and cleaning 3 times by using PBST; then GETV E H2 mab (1:1000) was added and incubated 2: 2H at room temperature, and washed 3 times with PBST; FITC-coat anti-mouse antibody (1:1000) was then added and incubated 1h at room temperature in the dark, after 3 washes with PBST, DAPI (1:1000) was added and incubated 10: 10 min at room temperature in the dark, and green fluorescence was observed on an inverted fluorescence microscope.

The results show (FIG. 2) that GETV-LA11 strain was recognized by GETV E-3H 2 mab and exhibited green fluorescence, whereas mock-infected cells did not.

Example 3 plaque identification of the strain LA11 of the cover-tower virus

Inoculating GETV-LA11 strain to monolayer Vero cells, incubating at 37deg.C for 1: 1h, discarding virus solution, replacing DMEM culture solution containing 1% methylcellulose, placing into incubator, culturing, discarding upper layer culture solution when infection 72 h has obvious plaque, staining with crystal violet, and observing and recording plaque morphology.

As shown in FIG. 3, the plaque morphology of GETV-LA11 strain on cells was significantly reduced, indicating that the replication capacity in vitro was reduced.

EXAMPLE 4 multistep growth curve of the Katavirus LA11 strain in BHK-21 cells

GETV-LA11 was inoculated into single-layered BHK-21 cells of 24-well plates at moi=0.01, after incubation at 37 ℃ for 1h, the virus solution was discarded, and washed 3 times with pre-chilled PBS, after which the DMEM medium containing 2% serum was replaced and placed in an incubator for further culture. Culture supernatants were collected at 12, 24, 36, 48, and 60 h, respectively, post-inoculation and virus titers in the culture supernatants were determined by TCID ₅₀ method.

The results show (FIG. 4) that GETV-LA11 proliferated efficiently and replicated to higher titers on BHK-21 cells.

Example 5 evaluation of safety of the strain LA11 of the Katavirus on susceptible animals

1) Pathogenicity experiment of Gaota virus LA11 strain on ICR neonatal rat

30 ICR neonatal rats of 2 days old, 10 in each group, were housed in separate cages by their parents. GETV-HN and GETV-LA11 were diluted to 10 ⁷TCID₅₀/mL and the 30 uL diluted viral fluid was inoculated subcutaneously. The control mice were inoculated subcutaneously with 30 uL sterile PBS. Mice were observed daily after inoculation and recorded for mortality and weight changes.

The results are shown in FIG. 5, and demonstrate that high doses GETV-LA11 vaccinated with the neonatal ICR rats did not cause death and significant weight loss, similar to the control results. The parent GETV-HN strain causes the rapid death of the suckling mice.

2) Pathogenicity experiment of Gatta Virus LA11 strain on newborn piglets

15 Newborn piglets without GETV antibodies were tested for evaluation of safety of LA11, 5 in each group. Group a was vaccinated with 1 first part (5 x 10 ⁷TCID₅₀ virus per first part) of GETV-HN strain P1 generation, group B was vaccinated with 1 first part (5 x 10 ⁷TCID₅₀ virus per first part) of LA11 strain, and group C served as a blank vaccination control. Serum viremia was monitored 1-3 days after challenge, and clinical symptoms and body temperature changes were observed daily until 14 days after inoculation.

The results are shown in Table 1. Obvious viremia appears 1-3 days after the GETV-HN strain infection group attacks the toxin, obvious clinical symptoms appear on all piglets, the symptoms are serious diarrhea, hind limb paralysis and emaciation, and all piglets die within 14 days after the toxin attack. All pigs in the LA11 strain infection group have no obvious clinical symptoms (such as diarrhea, camping, hind limb paralysis, emaciation and the like), the body temperature is not increased, and viremia cannot be detected on the 3 rd day. The piglets of the blank control group had no abnormality during the observation period. Serum from surviving animals was collected 2 weeks after challenge and tested for GETV p-E1 antibodies by ELISA. The results indicated that the LA11 group piglets had increased antibody levels 2 weeks after challenge, while the control pigs remained free of detectable antibodies (fig. 6).

The above results demonstrate that GETV-LA11 strain has high safety in susceptible ICR rats and newborn piglets and good immunogenicity in piglets.

Table 1 safety test piglet monitoring conditions

Example 6 immunoprotection efficacy test of the strain LA11 of the Katavirus on ICR mice

ICR mice at 3 weeks of age were randomly divided into 3 groups of 5 animals each. Wherein group A ICR mice were inoculated subcutaneously 100uL with the GETV-HN strain of 10 ⁵TCID₅₀), group B ICR mice were inoculated subcutaneously 100uL with the GETV-LA11 strain of 10 ⁵TCID₅₀), and group C ICR mice were inoculated subcutaneously 100uL of sterile PBS. Serum was collected 1-3 days after immunization for the determination of viremia after infection. All mice were challenged 14 days after immunization and inoculated subcutaneously with 100uL strain GETV-HN containing 10 ⁶TCID₅₀. Serum was collected daily 3 days after challenge for viremia monitoring, and mice were dissected at day 3 post challenge to detect spleen viral load.

The results show that mice in group LA11 have significantly increased antibody titers after immunization, and that mice in group LA11 also show the same complete protection as mice in group GETV-HN after challenge with the high-dose parental strain, with no detectable viremia on days 1-3, whereas the control group can detect viremia. In addition, no infectivity GETV was detected in the spleens of mice in group LA11 and GETV-HN groups on day 3 post challenge, whereas a high viral load of GETV was detected in the spleens of mice in the control group (fig. 7). Thus, these results indicate that LA11 is equally well immunogenic in 3 week old mice and protects mice from infection by the high dose parent GETV.

Example 7 test of the efficacy of the Ala 11 strain of Katavirus in passive immunoprotection against newborn piglets

3 Pregnant sows without GETV antibodies were tested for evaluation of the passive protective efficacy of LA 11. Group A2 gilts were injected with 1 part (each part having a virus content of 10 ⁵TCID₅₀) of LA11 strain 2 mL in neck muscle, and group B1 gilts were used as blank inoculation control to be inoculated with sterile physiological saline 2 mL.

After the sow produces piglets, the piglets are subjected to toxin eliminating, and 5 piglets are treated in each group. Each piglet was vaccinated with 1 first part (each with a virus content of 5 x 10 ⁷TCID₅₀) of GETV-HN strain P1 generation, clinical symptoms and body temperature changes were observed daily within 14 days after challenge, and serum viremia was monitored on days 1-3 after challenge, and the results are shown in table 2.

The results showed that the newborn piglets of the immunized sow group obtained GETV p-E1 antibody by sucking breast milk, whereas the newborn piglets of the control sow group had no detectable antibody before challenge. After the piglets of the LA11 strain immune sow group are detoxified, no obvious clinical symptoms appear in all the piglets, the body temperature is not increased, and no detectable viremia exists; the control sow group had clinical symptoms such as diarrhea, camping on lying, emaciation, and the like, wherein 2 piglets died (fig. 8), but the body temperature did not rise during the monitoring. In addition, high levels of serum viremia can be detected 1-3 days after challenge. The GETV-LA11 strain immunized pregnant sow has good passive immune protection effect on piglets, and can protect offspring from being infected by GETV with high dose. In addition, the GETV p-E1 antibodies of the LA11 strain immunized sow group piglets gradually decreased after 14 days of challenge, indicating that the antibodies transferred through colostrum are sufficient to achieve a sterilizing immune effect. Therefore, the LA11 strain is safe for pregnant sows under the immune dosage, and can completely protect the offspring of the strain from being infected by high dosage GETV through colostrum, thereby having good immune protection effect.

Table 2 monitoring of piglets in passive immunization test

Example 8 genetic stability test of the strain LA11 of the Katavirus

The GETV-LA 11 strain of P0-generation virus was serially passaged on BHK-21 cells at MOI=0.01. BHK-21 cells have higher susceptibility to GETV-LA 11 strain and faster lesions, and are used for vaccine virus propagation. Specifically: the GETV-LA 11 strain with moi=0.01 was inoculated into a monolayer of BHK-21 cells, incubated in an incubator at 37 ℃ for 1h, and then replaced with DMEM containing 2% serum. Continuing to culture 36-48 h, when 90% of cells are observed to have lesions, placing the cells in a refrigerator at-80 ℃ for freezing and thawing once. By analogy, passages were performed on BHK-21 cells for 20 consecutive passages, and F10 and F20 passages were used to extract RNA for amplifying nsP3 gene and E2 and E1 genes, respectively, and the stability of the deleted region and the virus glycoprotein gene was observed.

The results show that the sequence of the deletion region and the E2 and E1 genes are not changed when the conventional method is carried out on the BHK-21 cells, and the GETV-LA 11 strain is genetically stable on the BHK-21 cells, so that the BHK-21 cells can be used as cells for vaccine production.

The protection of the present invention is not limited to the above embodiments. Variations and advantages that would occur to one skilled in the art are included in the invention without departing from the spirit and scope of the inventive concept, and the scope of the invention is defined by the appended claims.

Claims (8)

1. A attenuated vaccine strain of the Kappaphycus alvarezii, named Kappaphycus alvarezii (Getah Virus) GETV-LA11, is preserved in China center for type culture collection, and has the preservation address of: chinese, wuhan, university of Wuhan, accession number: cctccc NO: v202442, the preservation time is 2024, 4, 9 days.

2. Use of the attenuated strain of katavirus of claim 1 in the preparation of a katavirus vaccine.

3. The use according to claim 2, wherein the vaccine is a live attenuated or inactivated vaccine against the head-tower virus.

4. The use according to claim 2, wherein the vaccine comprises the attenuated strain of the galta virus according to claim 1 as the only active ingredient or one of the active ingredients.

5. Use of a attenuated strain of katavirus according to claim 1 for the manufacture of a medicament for the prevention of a disease caused by katavirus.

6. A vaccine comprising the attenuated strain of the cover virus of claim 1.

7. The vaccine of claim 6, further comprising a pharmaceutically acceptable adjuvant.

8. Use of the vaccine of claim 6 or 7 for the manufacture of a medicament for the prevention of diseases caused by the getavirus.