The Construction and Immunogenicity Analyses of a Recombinant Pseudorabies Virus with Senecavirus A VP2 Protein Coexpression

ABSTRACT Senecavirus A (SVA)-associated porcine idiopathic vesicular disease (PIVD) and pseudorabies (PR) are highly contagious swine diseases that pose a significant threat to the swine industry in China. Since there is currently no effective commercial vaccine against SVA, the virus has spread widely throughout China and its pathogenicity has increased over the last decade. In this study, a recombinant strain named rPRV-XJ-ΔTK/gE/gI-VP2 was constructed by using the pseudorabies virus (PRV) variant strain XJ as the parental virus and by deleting the TK/gE/gI gene while coexpressing SVA VP2. The recombinant strain can stably proliferate and express foreign protein VP2 in BHK-21 cells while having a similar virion appearance to that of the parental strain. rPRV-XJ-ΔTK/gE/gI-VP2 is safe and effective for BALB/c mice, inducing high levels of neutralizing antibodies against both PRV and SVA, providing 100% protection from the virulent PRV strain. Histopathological examination and quantitative PCR (qPCR) assay have demonstrated that SVA can infect mice through intranasal inoculation, while the vaccination of mice with rPRV-XJ-ΔTK/gE/gI-VP2 can significantly reduce SVA virus copies and alleviate the pathological inflammatory changes in the heart and liver. The evaluation of the safety and immunogenicity indicates that rPRV-XJ-ΔTK/gE/gI-VP2 holds promise as a candidate vaccine against PRV and SVA. IMPORTANCE This study reports the construction of a recombinant PRV with SVA for the first time, and the resulting virus, rPRV-XJ-ΔTK/gE/gI-VP2, can induce high levels of neutralizing antibodies against both PRV and SVA in model mice. These findings provide valuable insights for evaluating the effectiveness of rPRV-XJ-ΔTK/gE/gI-VP2 as a vaccine for pigs. Additionally, this study reports transient SVA infection in mice, with qPCR assays showing that the copies of the SVA 3D gene peaked at 3 to 6 days postinfection and fell below the sensitivity threshold by 14 days postinfection. The copies of the gene were more regular and at a higher level in the heart, liver, spleen, and lung tissue.

In addition, many abbreviations are used without ever being defined or if they are defined, the reader needs to use the search function. Examples include but are not limited to lines 63, 149, and 274. 2. Abstract line 16 states that SVA VP2 activates a Th2 response to enhance the humoral response to SVA. I am not sure there is enough data to fully support this statement since Th1 cytokines like IFN gamma and IL2 are also produced. In the discussion, line 263, this conclusion is also made with the statement that IL-4 levels in the vaccine group was higher than the rPRV-XJ-deltaTK/gE/gI group. However, this was not a statistically significant difference. This is also a confusing sentence, because what does "vaccine group" refer to? rPRV-XJ-deltaTK/gE/gI is also a vaccine group, but I assume "vaccine group" refers to the vaccine expressing VP2. 3. In the animal experiments, is the vaccine virus shed and if so for how long? 4. Do the mice infected with Senecavirus exhibit any clinical symptoms from cardiac disease? What are the main clinical manifestations of Senecavirus in pigs and are they similar to mice? Further discussion of this could be helpful to see how well the mouse model recapitulates disease in the target population. Minor 1. Figure 4 does not clearly state the route of immunization and challenge. Searching the methods suggest intranasal immunization but stating this clearly in figure legend or in the section referring to the figure would be helpful. 2. The methods section, line 411, mention a "proportion method". What is this method? Also, the y-axis of Figure 4 shows neutralization titer in log base 2, but it is not clear what the numbers refer to? Is this the dilution of serum that leads to 50% plaque reduction?
Reviewer #2 (Comments for the Author): Article by Tao et al., Microbiology Spectrum Presented manuscript is devoted to the development of live attenuated virus vaccine against Senecavirus A (SVA) and pseudorabies virus (PRV), highly contagious swine diseases that present a substantial burden for agricultural industry. Despite its importance, presented study has several issues outlined below.
Major points: 1. Figure 2D: There is about one log difference in virus titer between parental strain and the recombinant virus at 0h postinfection. How is that possible if same infectious dose of 0.1 MOI was used to infect cells? How was virus infectivity assessed? It is not described in Methods! Was cell density different? The difference in virus titer remains stable throughout the experiment and at 48h post infection the titers of both viruses become almost identical. This suggests that the difference observed in titer is due to different starting dose rather than a result of deletion of 3 genes. I cannot agree with statement that kinetic of virus growth is different. Growth kinetics is reflected by curve slope and shape which apparently very similar for both viruses. However, formal analysis of curve slope and parallelism can provide a sound answer to this question. Methods section indicates that three viruses were used for growth kinetic experiment: PRV-XJ, rPRV-XJ-deltaTK and rPRV-deltaTK/gE/gI-VP2. rPRV-XJ-deltaTK is not shown on Figure 2D. What is the reason for not including this virus into the figure? There is no description how virus titer was measured. Methods only says that cells and supernatant were harvested. 2. How exactly size of plagues was measured? The technique is not described! Could different plaque size be a result of the virus dose? 3. Transmission electron microscopy is not described in Methods. 4. Histopathology technique is not described in Methods. 5. Is mice survival the only measure of safety of recombinant virus created? Apparently, deletion of gE, gI and TK affects virus virulence and transmissibility. As such, appropriate references should be provided and subsequently discussed. 6. The following statement requires a reference: "IFN-g can be used as a standard to evaluate the cell-mediated immune effects of vaccines; it plays an important role in innate and adaptive immunity". 7. Line 143-144: "the level of IFN-g improved significantly..." Was it reduced/diminished in the first place to be improved after vaccination? Did authors meant "increased"? 8. Authors need to provide a reason why ConA-induced stimulation was used as a comparison? 9. "IL-2 was secreted by Th1 cell, IL-4 was secreted by Th2 cell." This statement implies that authors investigated Th1 and Th2 cells defined by flow cytometry or other methods. It seems that this phrase is introductory to the following experiment where IL-2 and IL-4 serum levels were investigated. If this is the case, the sentence to be revised and supplemented with appropriate reference. 10. Lines 173-174: Complete lack of flow. It is absolutely unclear why data on SVA in mouse tissues are presented here? In the preceding paragraph, only immunization and infection with PRV is described! Where these mice infected with SVV also or it was a separate experiment? Experiment testing SVA infectivity and consequences of infection is not described raising concerns about study integrity. Moreover, there is a statement that it is not known if SVV can infect mice. However, simple search yields a reference doi:10.1016/j.rvsc.2021.12.010 providing -evidence that SVV in fact infects mice. 11. What is 3D RNA is not explain either. 12. Graphical representation of immunization/virus challenge experiment would be helpful to understand study design. Were both viruses given simultaneously, how much time was between immunization and virus challenge?
13. Discussion is poorly written, extensively long and not focused. Some if it, the first two paragraphs, for example, is a repeat of Introduction. Several following paragraphs is a simple repeat of the results observed. In lines 262-263 authors state: "while the IL-4 level in the recombinant vaccine group was higher than rPRV-XJ-ΔTK/gE/gI group". According to Figure 5D this difference was not statistically significant and authors admit that in the corresponding statement on lines 156-158. Therefore, the following implication of "improved" IL-4 levels via activation of TH2 cells and the following enhancement of humoral response seems speculative and is not supported by data provided. In contrast, presented results rather show no difference between IL-2 and IL-4 that is well in line with general understanding of immune response to viral infection that in fact immunization with recombinant virus is. Line 269: As mentioned before, there is published study about SVA infection in mice. This should be discussed in regards with data presented in this manuscript. Study limitations are not discussed. 14. Can the recombinant virus created in this study be transmitted between animals?  Figure 2B: what is Mock? Explanation in figure legend is needed. 5. Figure 2B: while beta-actin provides a control for cell proteins, another control for viral protein(s) would be helpful here. 6. Fig. 4A and 4B: What is the reason of showing results of anti-gB ELISA as signal-to-noise ratio and VP2 ELISA in absorbance units? 7. Fig.5A is not informative and rather shows raw data. It can be moved into Supplementary materials. 8. Fig. 6A, 6B and 6C replicate data provided in Fig.3. These data can be moved into Supplementary materials with reference in text that survival kinetic and brain tissue damage as well as serum IL-6 and TNF levels were similar to that found in safety experiment.
Staff Comments:

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Manuscript Number (Spectrum05229-22): The construction and immunogenicity analyses of a recombinant pseudorabies virus with
Senecavirus A VP2 protein co-expression

Dear Editor and Reviewers,
Thank you for giving us the opportunity to resubmit our revised manuscript. We appreciate the concerns and suggestions provided by the reviewers and editor, and we have revised our manuscript accordingly. The modified parts in the text were indicated with red color, and the responses to the reviewers are as follows. These suggestions helped us improve the manuscript, and we hope that you find it suitable for publication.
I look forward to hearing from you soon. In the discussion, line 263, this conclusion is also made with the statement that IL-4 levels in the vaccine group was higher than the rPRV-XJ-deltaTK/gE/gI group. However, this was not a statistically significant difference. This is also a confusing sentence, because what does "vaccine group" refer to? rPRV-XJ-deltaTK/gE/gI is also a vaccine group, but I assume "vaccine group" refers to the vaccine expressing VP2.  Orally challenged with 2×10 −7 TCID50 of SVA CH-HNCY-2019 can cause significant pathological changes in the heart, lung, liver, spleen, kidney, brain and duodenum tissue of mice(2)(line 277-281). Gene copies in the heart, liver, spleen, and lung tissues were more regular and had a higher level, consistent with the tropism of SVA for various organs in naturally infected newborn piglets(3) (line285-288). This result also provides support for mice to be used as animal models of SVA infection.
These modified contents have been marked with red color in DISCUSSION.
Minor concerns: 1. Figure  2. The methods section, line 411, mention a "proportion method". What is this method?
Also, the y-axis of Figure 4 shows neutralization titer in log base 2, but it is not clear what the numbers refer to? Is this the dilution of serum that leads to 50% plaque reduction?
Response: Thanks for your question. The serum neutralization titer was measured by plaque reduction assay, the serum dilution that reduced the number of plaques by 50% was used as the neutralization titer for this serum sample. Proportion was used as the calculation method of plaque reduction assay. For example, the percentage of plaque reduction of the serum sample under the serum dilution at 2 4 is 53.85%, the percentage of plaque reduction at the serum dilution at 2 5  However, formal analysis of curve slope and parallelism can provide a sound answer to this question. Methods section indicates that three viruses were used for growth kinetic experiment: PRV-XJ, rPRV-XJ-deltaTK and rPRV-deltaTK/gE/gI-VP2.
rPRV-XJ-deltaTK is not shown on Figure 2D. What is the reason for not including this virus into the figure? There is no description how virus titer was measured.

Methods only says that cells and supernatant were harvested.
Response: Thanks for your question. Sorry for my carelessness, the replication kinetics of rPRV-XJ-ΔTK/gE/gI-VP2 has been re-tested, and the virus one-step growth curve of rPRV-XJ-ΔTK/gE/gI has been added into Figure 2D. In addition, the plaque assays of rPRV-XJ-ΔTK/gE/gI has been added into Figure 2E  Apparently, deletion of gE, gI and TK affects virus virulence and transmissibility. As such, appropriate references should be provided and subsequently discussed.

Response:
Thanks for your question. In this study, the serum levels of inflammatory cytokines and the brain tissue histopathology assay of the mice vaccinated with rPRV-XJ-ΔTK/gE/gI-VP2 also were used as the measure of safety assessment.
Appropriate references have been provided and subsequently discussed. The modified content has been marked with red color in DISCUSSION (line 245-251).

Comment 6:
The following statement requires a reference: "IFN-g can be used as a standard to evaluate the cell-mediated immune effects of vaccines; it plays an important role in innate and adaptive immunity".
Response: Thanks for your question. This sentence has been modified and marked with red color: Interferon-γ (IFN-γ) can serve as a criterion for evaluating the specific T cell-mediated immune effects of vaccines (4) (line 153-154).  Response: Thanks for your question. Con A is a phytohemagglutinin with potent mitogenic capacity, which can be used as a positive standard for stimulating splenic lymphocyte proliferation (5). This sentence has been added at line 162-163 and marked with red color.  In lines 262-263 authors state: "while the IL-4 level in the recombinant vaccine group was higher than rPRV-XJ-ΔTK/gE/gI group". According to Figure 5D this difference was not statistically significant and authors admit that in the corresponding statement on lines 156-158. Therefore, the following implication of "improved" IL-4 levels via activation of Th2 cells and the following enhancement of humoral response seems speculative and is not supported by data provided. In contrast, presented results rather show no difference between IL-2 and IL-4 that is well in line with general understanding of immune response to viral infection that in fact immunization with recombinant virus is.
Line 269: As mentioned before, there is published study about SVA infection in mice.
This should be discussed in regards with data presented in this manuscript.
Study limitations are not discussed. Response: Thanks for your question. We apologize for the limitations of our experimental design. This study did not include experiments on the transmission of the recombinant virus between animals. Although we believe that it has the potential to be transmitted from animal to animal, further experimental verification is required to confirm this.

Minor points:
1. Editorial help with English language is required.  Figure 2B: what is Mock? Explanation in figure legend is needed.

Response:
Thanks for your question. The explanation of Mock has been added into the figure legend (line 688) and marked with red color. 5. Figure 2B: while beta-actin provides a control for cell proteins, another control for viral protein(s) would be helpful here.  Fig.5A is not informative and rather shows raw data. It can be moved into Supplementary materials.

Response:
Thanks for your question. So sorry for cannot following this suggestion.
Including the Flow cytometry (FCM) scatter plot in the main text can certainly provide a clearer understanding of the experimental results, and it is common practice in many cellular immunity-related articles (8)(9)(10)(11). So, in this study, we prefer to put 8. Fig. 6A, 6B and 6C replicate data provided in Fig.3. These data can be moved into Supplementary materials with reference in text that survival kinetic and brain tissue damage as well as serum IL-6 and TNF levels were similar to that found in safety experiment.
Response: Thanks for your question. So sorry for cannot following this suggestion.