A HSV1 mutant leads to an attenuated phenotype and induces immunity with a protective effect

Herpes simplex virus type 1 (HSV1) is a complicated structural agent with a sophisticated transcription process and a high infection rate. A vaccine against HSV1 is urgently needed. As multiple viral-encoded proteins, including structural and nonstructural proteins, contribute to immune response stimulation, an attenuated or deficient HSV1 vaccine may be relatively reliable. Advances in genomic modification technologies provide reliable means of constructing various HSV vaccine candidates. Based on our previous work, an M6 mutant with mutations in the UL7, UL41, LAT, Us3, Us11 and Us12 genes was established. The mutant exhibited low proliferation in cells and an attenuated phenotype in an animal model. Furthermore, in mice and rhesus monkeys, the mutant can induce remarkable serum neutralizing antibody titers and T cell activation and protect against HSV1 challenge by impeding viral replication, dissemination and pathogenesis.

The significant weekness of this study is that while M6 induces neutralizing antibodies, and reduce the viral replication, the level of neutralizing antibody titer is very low, and M6 does not prevent virus replication in the tissues, and latency establishment in sensory ganglia. The concludion drawn from this study is not in the accordence with the data presented.
R: Thank you for the summary and comments. In our revised manuscript, we have added some experiments. The immunogenicity of M6 was evaluated in rhesus macaques via detection of their neutralizing antibody response and T cell response in a dose-dependent immunization test via the intramuscular route ( Fig 5A).The results showed that immunizing monkey with three doses can induce significant proliferation of CD4+ and CD8+ T cells with IFN-γ specificity (Figs 5B and 5C).
Then, the immunogenicity and protective effect of M6 were further investigated in a challenge test with the medium dose ( Fig 6A). Although the neutralizing antibody titer is still low, theseimmunized animals were asymptomaticafter viral challenge, while all of the animals in the positive control group developed oral vesicles or exhibited redness around the eyes after WT strain challenge (Fig 6C).
Histopathological detection also indicated the protectiveefficiency of M6.
For the latency establishment, we have no related experiments in this manuscript. R: Thank you for the summary. We have added the experiments to furtherstrengthen their findings and conclusionsaccording to your suggestions.

Part II-Major Issues: Key Experiments Required for Acceptance
Reviewer #1: No new experiments are suggested, because additional experiment will not make this manuscript better.
Reviewer #2: 1. In addition to detecting neutral antibodies and responsive T cell proliferation in mice or rhesus monkeys, CD4+ and CD8+ T cell responses and of cytokines expression should also be detected by flow cytometry.
R: This suggestion is very helpful for us. In the newly added dose-dependent inoculation experiment, CD4+ and CD8+ T cell responses and IFN-γ expression were detected by flow cytometry at 7, 14 and 28 days post immunization. The results showed that M6 immunization could induce the significant increase the numbers of CD4 + T IFN-γ + and CD8 + T IFN-γ + in both the mouse and rhesus monkey models. We have added these results to the Results section. These results are similar to the ELISpot results we used in the manuscript, which indicated that ELISpot assay is another available method for detecting the level of IFN-γ-producing lymphocytes.

The authors should take M6 inoculation in mice and rhesus monkeys in a dose-dependent manner to examine indicators of specific immune responses.
R: Thank youfor this useful suggestion. We inoculated the mice with three doses (2x10 3 , 1x10 4 or 5x10 4 pfu) and the rhesus monkeys with three doses (2x10 4 , 1x10 5 or 5x10 5 CCID50) of M6. Moreover, we detected the neutralizing antibodies and responsive T cell proliferation in mice or rhesus monkeys. The results indicate that M6 immunization in three doses could induce a significant increase in the numbers of CD4+T IFN-γ+ and CD8+T IFN-γ+ in both the mouse and rhesus monkey models at 7, 14 and 28 days post immunization. However, at 28 days post immunization, we detectedonly low neutralizing antibody titers in the mouse and rhesus monkey models. We have added these results to the Results section. genome could be simultaneously modified with high efficiency and with no off-target modifications. In our manuscript, we designed specific primers surrounding and inside the mutated regions of theUs3, Us11 and Us12 genes. The PCR assays using these primers indicated that the double copies of theUs3, Us11 and Us12 genes were modified (S1 Fig). We have added these results to the Results section.

Part III-Minor Issues: Editorial and Data Presentation Modifications
Reviewer #1: 1.The title of this manuscript suggests that M6 mutant induces immunity with a protective effect against viral infection in mice and rhesus macaques; however, the data clearly shows that it does not prevents viral infection in mice or rhesus.
R: Based on the results in mice and rhesus monkeys, the M6 mutant induces remarkable serum neutralizing antibody titers and T cell activation and protects against HSV1 challenge by impeding viral replication, dissemination and pathogenesis.
Thus, we can state that the M6 mutant can prevent viral infection in mice or rhesus monkeys.

Many sections of figures lack the titles or the details on the y axis.
R: We have modified the figures by adding more details tothe y-axis.

In the introduction, authors stated that HSV-1 cause occasional genital herpes infection. Epidemiology indicates that more that 50% of first-time genital herpes infections are now caused by HSV-1 virus.
R: We have checked the relevant reference and revised the sentences in the introduction section.

Reviewer #2: None
Reviewer #3: 1.In Figure 1C showed that M6 immunization could induce a significant increase in the numbers of CD4 + T IFN-γ + and CD8 + T IFN-γ + in both the mouse and rhesus monkey models. We have added these results to the Results section. These results are similar to the ELISpot results we used in the manuscript, which indicated that ELISpot assay is also an available method for detecting the level of IFN-γ producing lymphocytes. Figure 4 and R:In our other experiments (unpublished data), we confirmed using two-dimensional electrophoresis (2-DE) and mass spectrometry that the serum antibodies of HSV1-positive patients mainly recognized capsid proteins, such as proteins encoded by UL26.5 and UL42 genes. Meanwhile, the membrane structural glycoproteins, which are the main immunogens recognized by neutralizing antibodies, were not detected. It is suggested that these encoded capsid proteins are likely to be the dominant antigens that interact with immune response and stimulate the immune system to produce antibodies during the HSV1 infection. Based on these results, we think that the neutralizing antibody titer cannot be used as aunique factor to evaluate viral immunogenicity. In our manuscript, there was no difference in neutralizing antibody titers between M3-and M6-immunized mice;however, we modified more genes involved in the apoptosis and antigen presentation of the M6 mutant, and M6 may be safer than the M3 strain.