Purging viral latency by a bifunctional HSV-vectored therapeutic vaccine in chronically SIV-infected macaques

  1. Ziyu Wen
  2. Pingchao Li
  3. Yue Yuan
  4. Congcong Wang
  5. Minchao Li
  6. Haohang Wang
  7. Minjuan Shi
  8. Yizi He
  9. Mingting Cui
  10. Ling Chen
  11. Caijun Sun

  • Curated by eLife

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    eLife Assessment

    In this useful study, the authors tested a novel approach to eradicate the HIV reservoir by constructing a herpes simplex virus (HSV)-based therapeutic vaccine designed to reactivate HIV from latently infected cells and induce an immune response to kill such infected cells. Testing this approach with SIV in a primate model, the authors report that the SIV reservoir was reduced. However, the evidence presented appears to be incomplete because the animal group size was small and the SIV reservoir size highly variable.

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Abstract

The persistence of latent viral reservoirs remains the major obstacle to eradicating human immunodeficiency virus (HIV). We herein found that ICP34.5 can act as an antagonistic factor for the reactivation of HIV latency by herpes simplex virus type I (HSV-1), and thus recombinant HSV-1 with ICP34.5 deletion could more effectively reactivate HIV latency than its wild-type counterpart. Mechanistically, HSV-ΔICP34.5 promoted the phosphorylation of HSF1 by decreasing the recruitment of protein phosphatase 1 (PP1α), thus effectively binding to the HIV LTR to reactivate the latent reservoirs. In addition, HSV-ΔICP34.5 enhanced the phosphorylation of IKKα/β through the degradation of IκBα, leading to p65 accumulation in the nucleus to elicit NF-κB pathway-dependent reactivation of HIV latency. Then, we constructed the recombinant HSV-ΔICP34.5 expressing simian immunodeficiency virus (SIV) env, gag, or the fusion antigen sPD1-SIVgag as a therapeutic vaccine, aiming to achieve a functional cure by simultaneously reactivating viral latency and eliciting antigen-specific immune responses. Results showed that these constructs effectively elicited SIV-specific immune responses, reactivated SIV latency, and delayed viral rebound after the interruption of antiretroviral therapy (ART) in chronically SIV-infected rhesus macaques. Collectively, these findings provide insights into the rational design of HSV-vectored therapeutic strategies for pursuing an HIV functional cure.

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  1. Version published to 10.7554/elife.95964.4 on eLife
  2. Version published to 10.7554/elife.95964 on eLife
  3. Version published to 10.7554/elife.95964.3 on eLife
  4. eLife

    eLife Assessment

    In this useful study, the authors tested a novel approach to eradicate the HIV reservoir by constructing a herpes simplex virus (HSV)-based therapeutic vaccine designed to reactivate HIV from latently infected cells and induce an immune response to kill such infected cells. Testing this approach with SIV in a primate model, the authors report that the SIV reservoir was reduced. However, the evidence presented appears to be incomplete because the animal group size was small and the SIV reservoir size highly variable.

  5. eLife

    Reviewer #1 (Public review):

    Summary:

    Authors constructed a novel HSV-based therapeutic vaccine to cure SIV in a primate model. The novel HSV vector is deleted for ICP34.5. Evidence is given that this protein blocks HIV reactivation by interference with the NFkappaB pathway. The deleted construct supposedly would reactivate SIV from latency. The SIV genes carried by the vector ought to elicit a strong immune response. Together the HSV vector would elicit a shock and kill effect. This is tested in a primate model.

    Strengths and weaknesses:

    (1) Deleting ICP34.5 from the HSV construct has a very strong effect on HIV reactivation. The mechanism underlying increased activation by deleting ICP34.5 is only partially explored. Overexpression of ICP34.5 has a much smaller effect (reduction in reactivation) than deletion of ICP34.5 (strong activation); this is acknowledged by the authors that no full mechanistic explanation can be given at this moment.

    (2) No toxicity data are given for deleting ICP34.5. How specific is the effect for HIV reactivation? A RNA seq analysis is required to show the effect on cellular genes.

    A RNA seq analysis was done in the revised manuscript comparing the effect of HSV-1 and deleted vector in J-LAT cells (Fig S5). More than 2000 genes are upregulated after transduction with the modified vector in comparison with the WT vector. Hence, the specificity of upregulation of SIV genes is questioned. Authors do NOT comment on these findings. In my view it questions the utility of this approach.

    (3) The primate groups are too small and the results to variable to make averages. In Fig 5, the group with ART and saline has two slow rebounders. It is not correct to average those with the single quick rebounder. Here the interpretation is NOT supported by the data.

    Although authors provided some promising SIV DNA data, no additional animals were added. Groups of 3 animals are too small to make any conclusion, especially since the huge variability in response. The average numbers out of 3 are still presented in the paper, which is not proper science.

    No data are given of the effect of the deletion in primates. Now the deleted construct is compared with an empty vector containing no SIV genes. Authors provide new data in Fig S2 on the comparison of WT and modified vector in cells from PLWH, but data are not that convincing. A significant difference in reactivation is seen for LTR in only 2/4 donors and in Gag in 3/4 donors. (Additional question what is meaning of LTR mRNA, do authors relate to genomic RNA??)

    Discussion

    HSV vectors are mainly used in cancer treatment partially due to induced inflammation. Whether these are suitable to cure PLWH without major symptoms is a bit questionable to me and should at least be argued for.

    The RNA seq data add on to this worry and should at least be discussed.

    Comments on revisions:

    The authors accept the limitations of the primate study (too small for strong conclusions). The new way of presenting the data clearly shows these limitations.

  6. eLife

    Author response:

    The following is the authors’ response to the previous reviews.

    Public Reviews:

    Reviewer #1 (Public review):

    Summary:

    The authors constructed a novel HSV-based therapeutic vaccine to cure SIV in a primate model. The novel HSV vector is deleted for ICP34.5. Evidence is given that this protein blocks HIV reactivation by interference with the NFkappaB pathway. The deleted construct supposedly would reactivate SIV from latency. The SIV genes carried by the vector ought to elicit a strong immune response. Together the HSV vector would elicit a shock and kill effect. This is tested in a primate model.

    Strengths and weaknesses:

    (1) Deleting ICP34.5 from the HSV construct has a very strong effect on HIV reactivation. The mechanism underlying increased activation by deleting ICP34.5 is only partially explored. Overexpression of ICP34.5 has a much smaller effect (reduction in reactivation) than deletion of ICP34.5 (strong activation); this is acknowledged by the authors that no full mechanistic explanation can be given at this moment.

    Thank you for your comments. We agree with you that the mechanism underlying increased reactivation by deleting ICP34.5 is only partially explored. As you pointed out, the deletion of ICP34.5 leads to a significant reactivation, while the overexpression of ICP34.5 has a relatively weak inhibitory effect on reactivation. This difference prompts us to further contemplate the role of HSV-1 in regulating HIV latency and reactivation. Our data (Figure S4), along with previous literature (Mosca et al., 1987, Nabel et al., 1988), have indicated that the ICP0 protein might play a crucial role in the reactivation of HIV latency. However, we found for the first time that ICP34.5 can play an antagonistic role with this reactivation. This is a very interesting topic for understanding the complicated interactions between host cells and different viruses. We will investigate the deeper insights in future studies, and we have mentioned this limitation in the revised Discussion Section. Thank you!

    (2) No toxicity data are given for deleting ICP34.5. How specific is the effect for HIV reactivation? A RNA seq analysis is required to show the effect on cellular genes.

    A RNA seq analysis was done in the revised manuscript comparing the effect of HSV-1 and deleted vector in J-LAT cells (Fig S5). More than 2000 genes are upregulated after transduction with the modified vector in comparison with the WT vector. Hence, the specificity of upregulation of SIV genes is questioned. Authors do NOT comment on these findings. In my view it questions the utility of this approach.

    Thank you for your mentions.

    (1) As for the toxicity of HSV-ΔICP34.5, it is well known that ICP34.5 is a neurotoxicity factor that can antagonize host immune responses, and thus deleting ICP34.5 is beneficial to improve the safety of HSV-based constructs. As expected, we have demonstrated experimentally that HSV-DICP34.5 exhibited lower virulence and replication ability than wild-type HSV-1 (Figure S1). Importantly, we also observed a significant decrease in the expression of inflammatory factors in PWLH when compared to wild-type HSV-1 (Figure 1I-K). These data suggested that the safety of HSV-DICP34.5 should be more tolerable than wild-type HSV vector.

    (2) The RNASeq analysis is aimed to explore the HSV-ΔICP34.5-induced signaling pathways, but it is not suitable to use this data for assessing the toxicity of HSV-ΔICP34.5 constructs. As for the RNASeq data, we think it is reasonable to observe many upregulated genes (which are involved in a variety of signaling pathways), since HSV-DICP34.5 constructs reactivated HIV latency more effectively than wild-type HSV by modulating the IKKα/β-NF-kB pathway and PP1-HSF1 pathway.

    (3) To further validate whether HSV-ΔICP34.5 can specifically activate the HIV latent reservoir, we conducted additional experiments using vaccinia virus and adenovirus as controls, and results showed that both vaccinia virus and adenovirus cannot effectively reactivate HIV latency (Figure S3). Moreover, the deletion of ICP0 gene from HSV-1 diminished the reactivation effect of HIV latency by HSV-1, and overexpressing ICP0 greatly reactivate the latent HIV (Figure S4, Figure S5), implying that this reactivation should be virus-specific and ICP0 plays an important factor on reversing HIV latency. Interestingly, we herein found that ICP34.5 can act as an antagonistic factor for this reactivation of HIV latency by HSV-1. Thus, after the deletion of ICP34.5, the ability of HSV to reverse HIV latency was significantly enhanced. Our research group will investigate the underlying mechanism in future studies. Thank you for your insightful mention.

    (3) The primate groups are too small and the results to variable to make averages. In Fig 5, the group with ART and saline has two slow rebounders. It is not correct to average those with the single quick rebounder. Here the interpretation is NOT supported by the data.

    Although authors provided some promising SIV DNA data, no additional animals were added. Groups of 3 animals are too small to make any conclusion, especially since the huge variability in response. The average numbers out of 3 are still presented in the paper, which is not proper science.

    No data are given of the effect of the deletion in primates. Now the deleted construct is compared with an empty vector containing no SIV genes. Authors provide new data in Fig S2 on the comparison of WT and modified vector in cells from PLWH, but data are not that convincing. A significant difference in reactivation is seen for LTR in only 2/4 donors and in Gag in 3/4 donors. (Additional question what is meaning of LTR mRNA, do authors relate to genomic RNA??)

    Thank you for your serious review and kind reminder.

    (1) We agree with you that it is not appropriated to use averages for this pilot study with limited numbers of macaques. We are currently unable to conduct another experiment with a larger number of macaques, but we think the results of this pilot study were very promising for further studies. Now, following your kind suggestions, we have removed the averages and now presented the data for each monkey individually in the revised manuscript. We have also modified the corresponding description accordingly (Line 254 to 262). Thank you for your understanding.

    (2) Regarding your comment about the lack of data on the deletion of ICP34.5 from HSV-1, we are sorry for previously unclear description. In fact, the empty vector used in our animal experiments not only does not contain SIV antigens but also has the ICP34.5 deletion. We have revised the corresponding description accordingly (For example, we use HSV-DICP34.5DICP47-empty, HSV-DICP34.5DICP47-sPD1-SIVgag/SIVenv instead of HSV-empty, HSV-sPD1-SIVgag/SIVenv). We hope this revision will address your question.

    (3) As for the reactivation effects observed in PLWH samples, the data may be not perfect, but we think this result (a significant difference in reactivation is seen for LTR in 2/4 donors and for Gag in 3/4 donors, and the purpose of detecting LTR RNA is to evaluate the level of virus replication) is promising to support our conclusion (The enhanced reactivation effect in primary CD4+ T cells by HSV-∆ICP34.5 than wild-type HSV). Of course, we recognize the need for more samples to gain a comprehensive understanding of reactivation effect in different individuals in future study. In addition, we corrected the description of LTR RNA (Lines 99-106 and 115-116). Thank you for the reminder!

    Discussion

    HSV vectors are mainly used in cancer treatment partially due to induced inflammation. Whether these are suitable to cure PLWH without major symptoms is a bit questionable to me and should at least be argued for.

    The RNA seq data add on to this worry and should at least be discussed.

    Thank you for your mention. As mentioned above, the RNASeq analysis is aimed to explore the HSV-ΔICP34.5-induced signaling pathways, but it is not suitable to use this data for assessing the toxicity of HSV-ΔICP34.5 constructs. Actually, ICP34.5 is a neurotoxicity factor that can antagonize innate immune responses, and thus ICP34.5 deletion is beneficial to improve the safety of HSV-based constructs. As expected, our data have demonstrated experimentally that HSV-DICP34.5 exhibited lower virulence and replication ability than wild-type HSV-1 (Figure S1). Importantly, HSV-DICP34.5 induced a lower level of inflammatory cytokines (including IL-6, IL-1β, and TNF-α) in primary CD4+ T cells from PLWH compared to HSV stimulation, likely due to its lower virulence and replication ability (Figure 1I-K). In addition, the CD4+ /CD8+ T cell ratio (Figure 5H) and body weight (Figure S10) after treatment were effectively ameliorated in the SIV-infected macaques of the ART+HSV-DICP34.5DICP47-sPD1-SIVgag/SIVenv group. Our data also demonstrated that there was no significant effect on the cell composition of peripheral blood in the SIV-infected macaques of ART+HSV-DICP34.5DICP47-sPD1-SIVgag/SIVenv group (Figure S11). These data suggested that the safety of HSV-DICP34.5 should be more tolerable than wild-type HSV vector. We have added a more comprehensive description in the revised Discussion (Lines 328-334). Thank you again for all of your kind comments and suggestions.

    Reviewer #2 (Public review):

    Summary:

    In this article Wen et. al., describe the development of a 'proof-of-concept' bi-functional vector based out of HSV-deltaICP-34.5's ability to purge latent HIV-1 and SIV genomes from cells. They show that co-infection of latent J-lat T-cell lines with a HSV-deltaICP-34.5 vector can reactivate HIV-1 from a latent state. Over- or stable expression of ICP 34.5 ORF in these cells can arrest latent HIV-1 genomes from transcription, even in the presence of latency reversal agents. ICP34.5 can co-IP with- and de-phosphorylate IKKa/b to block its interaction with NF-k/B transcription factor. Additionally, ICP34.5 can interact with HSF1 which was identified by mass-spec. Thus, the authors propose that the latency reversal effect of HSV-deltaICP-34.5 in co-infected JLat cells is due to modulatory effects on the IKKa/b-NF-kB and PP1-HSF-1 pathway.

    Next the authors cleverly construct a bifunctional HSV based vector with deleted ICP34.5 and 47 ORFs to purge latency and avoid immunological refluxes, and additionally expand the application of this construct as a vaccine by introducing SIV genes. They use this 'vaccine' in mouse models and show the expected SIV-immune responses. Experiments in rhesus macaques (RM), further elicit potential for their approach to reactivate SIV genomes and at the same time block their replication by antibodies. What was interesting in the SIV experiments is that the dual-functional vector vaccine containing sPD1- and SIV Gag/Env ORFs effectively delayed SIV rebound in RMs and in some cases almost neutralized viral DNA copy detection in serum. Very promising indeed, however there are some questions I wish the authors explored to answer, detailed below.

    Overall, this is an elegant and timely work demonstrating the feasibility of reducing virus rebound in animals, and potentially expand to clinical studies. The work was well written, and sections were clearly discussed.

    Strengths:

    The work is well designed, rationale explained and written very clearly for lay readers.

    Claims are adequately supported by evidence and well designed experiments including controls.

    We appreciate your positive comment for our work.

    Weaknesses:

    (1) It looks like ICP0 is also involved in latency reversal effects. More follow-up work will be required to test if this is in fact true.

    Both our data (Figure S4, Figure S5) and previous literature (Nabel et al., 1988, Mosca et al., 1987) have reported that HSV ICP0 may play a role in reversing HIV latency. However, the exact mechanisms behind this effect have not yet been fully elucidated. Of note, we herein reported for the first time that ICP34.5 can act as an antagonistic factor for this reactivation of HIV latency by HSV-1. Thus, after the deletion of ICP34.5, the ability of HSV to reverse HIV latency was significantly enhanced. Our research group will investigate the underlying mechanism in future studies. Thank you for your insightful mention.

    (2) It is difficult to estimate the depletion of the latent viral reservoir. The authors have tried to address this issue. A more convincing argument to this reviewer will be data to demonstrate that after the bi-functional vaccine, the animals show overall reduction in the number of circulating latent cells. The feasibility to obtain such a result is not clearly demonstrated.

    Thank you for your comment. As you mentioned, we have indeed measured both total DNA and integrated DNA (iDNA) in blood cells (see Figure 5E-F), which can provide support for the reduction of the latent viral reservoir. Thank you for your kind reminder.

    (3) The authors state that the reduced virus rebound detected following bi-functional vaccine delivery is due to latent genomes becoming activated and steady-state neutralization of these viruses by antibody response. This needs to be demonstrated. Perhaps cell-culture experiments from specimen taken from animals might help address this issue. In lab cultures one could create environments without antibody responses, under these conditions one would expect higher level of viral loads being released in response to the vaccine in question.

    Thank you for your valuable suggestion. We believe that the reduced virus rebound observed may be influenced by immune responses from T cells and antibodies induced by both ART and the vaccine. We appreciate your insight and agree that future studies should focus on investigating the activation effects of the vaccine under controlled conditions that simulate the absence of immune responses in primary animal cells. This will help us better understand the mechanisms involved and address your concerns more comprehensively.

    Reviewer #2 (Recommendations for the authors):

    The Authors have sufficiently addressed my comments. Below are a few minor changes that can help with clarity.

    Lines 126-127: This sentence should be changed. Perhaps, "these data suggests that .... Safety of... in PLWH might be tolerable, at least in vitro."

    Thanks for your suggestion. We have revised it accordingly. (Line 130).

    Lines 128-132: Would this not mean that reactivation is due to ICP0 gene? Have the authors tried to express ICP0-gene into J-Lat cells and see if that is the reason for reactivation? This seems somewhat incomplete. At the end of 132, please add ", in the presence of ICP0". Also a sentence describing this effect is warranted.

    Thank you for your insightful suggestion. Yes, both our data and previous literature supported that the ICP0 gene can play a significant role in the reactivation of HIV latency (Figure S4, Figure S5). Of note, we herein reported for the first time that ICP34.5 can act as an antagonistic factor for this reactivation of HIV latency by HSV-1. Thus, after the deletion of ICP34.5, the ability of HSV to reverse HIV latency was significantly enhanced. We have described this effect in the revised version accordingly. Additionally, we have added the phrase “in the presence of ICP0” to the results section (Lines 137) to clarify this point.

    MOSCA, J. D., BEDNARIK, D. P., RAJ, N. B., ROSEN, C. A., SODROSKI, J. G., HASELTINE, W. A., HAYWARD, G. S. & PITHA, P. M. 1987. Activation of human immunodeficiency virus by herpesvirus infection: identification of a region within the long terminal repeat that responds to a trans-acting factor encoded by herpes simplex virus 1. Proc Natl Acad Sci U S A 84: 7408.DOI: https://doi.org/10.1073/pnas.84.21.7408, PMID: 2823260

    NABEL, G. J., RICE, S. A., KNIPE, D. M. & BALTIMORE, D. 1988. Alternative mechanisms for activation of human immunodeficiency virus enhancer in T cells. Science 239: 1299.DOI: https://doi.org/10.1126/science.2830675, PMID: 2830675

  7. Version published to 10.7554/elife.95964.2 on eLife
  8. Version published to 10.1101/2024.02.20.581122v4 on bioRxiv
  9. Version published to 10.1101/2024.02.20.581122v3 on bioRxiv
  10. eLife

    eLife Assessment

    In this useful study, the authors tested a novel approach to eradicating HIV reservoirs by constructing a herpes simplex virus (HSV)-based therapeutic vaccine and evaluating efficacy in experimental infections of chronically SIV-infected, antiretroviral therapy (ART)-treated macaques. While mean viremia at rebound was lower in the HSV vaccine-treated group, the evidence presented appears to be incomplete, as the group size was small and the viral load at rebound was highly variable. This is a revised paper, but the support for the conclusions, particularly the effect of the HSV-vectored therapeutic vaccine on the SIV reservoir in the SIV-infected macaques, remains limited.

  11. eLife

    Reviewer #1 (Public review):

    Summary:

    The authors constructed a novel HSV-based therapeutic vaccine to cure SIV in a primate model. The novel HSV vector is deleted for ICP34.5. Evidence is given that this protein blocks HIV reactivation by interference with the NFkappaB pathway. The deleted construct supposedly would reactivate SIV from latency. The SIV genes carried by the vector ought to elicit a strong immune response. Together the HSV vector would elicit a shock and kill effect. This is tested in a primate model.

    Strengths and weaknesses:

    (1) Deleting ICP34.5 from the HSV construct has a very strong effect on HIV reactivation. The mechanism underlying increased activation by deleting ICP34.5 is only partially explored. Overexpression of ICP34.5 has a much smaller effect (reduction in reactivation) than deletion of ICP34.5 (strong activation); this is acknowledged by the authors that no full mechanistic explanation can be given at this moment.

    (2) No toxicity data are given for deleting ICP34.5. How specific is the effect for HIV reactivation? A RNA seq analysis is required to show the effect on cellular genes.

    A RNA seq analysis was done in the revised manuscript comparing the effect of HSV-1 and deleted vector in J-LAT cells (Fig S5). More than 2000 genes are upregulated after transduction with the modified vector in comparison with the WT vector. Hence, the specificity of upregulation of SIV genes is questioned. Authors do NOT comment on these findings. In my view it questions the utility of this approach.

    (3) The primate groups are too small and the results to variable to make averages. In Fig 5, the group with ART and saline has two slow rebounders. It is not correct to average those with the single quick rebounder. Here the interpretation is NOT supported by the data.

    Although authors provided some promising SIV DNA data, no additional animals were added. Groups of 3 animals are too small to make any conclusion, especially since the huge variability in response. The average numbers out of 3 are still presented in the paper, which is not proper science.

    No data are given of the effect of the deletion in primates. Now the deleted construct is compared with an empty vector containing no SIV genes. Authors provide new data in Fig S2 on the comparison of WT and modified vector in cells from PLWH, but data are not that convincing. A significant difference in reactivation is seen for LTR in only 2/4 donors and in Gag in 3/4 donors. (Additional question what is meaning of LTR mRNA, do authors relate to genomic RNA??)

    Discussion

    HSV vectors are mainly used in cancer treatment partially due to induced inflammation. Whether these are suitable to cure PLWH without major symptoms is a bit questionable to me and should at least be argued for.

    The RNA seq data add on to this worry and should at least be discussed.

  12. eLife

    Reviewer #2 (Public review):

    Summary:

    In this article Wen et. al., describe the development of a 'proof-of-concept' bi-functional vector based out of HSV-deltaICP-34.5's ability to purge latent HIV-1 and SIV genomes from cells. They show that co-infection of latent J-lat T-cell lines with a HSV-deltaICP-34.5 vector can reactivate HIV-1 from a latent state. Over- or stable expression of ICP 34.5 ORF in these cells can arrest latent HIV-1 genomes from transcription, even in the presence of latency reversal agents. ICP34.5 can co-IP with- and de-phosphorylate IKKa/b to block its interaction with NF-k/B transcription factor. Additionally, ICP34.5 can interact with HSF1 which was identified by mass-spec. Thus, the authors propose that the latency reversal effect of HSV-deltaICP-34.5 in co-infected JLat cells is due to modulatory effects on the IKKa/b-NF-kB and PP1-HSF-1 pathway.

    Next the authors cleverly construct a bifunctional HSV based vector with deleted ICP34.5 and 47 ORFs to purge latency and avoid immunological refluxes, and additionally expand the application of this construct as a vaccine by introducing SIV genes. They use this 'vaccine' in mouse models and show the expected SIV-immune responses. Experiments in rhesus macaques (RM), further elicit potential for their approach to reactivate SIV genomes and at the same time block their replication by antibodies. What was interesting in the SIV experiments is that the dual-functional vector vaccine containing sPD1- and SIV Gag/Env ORFs effectively delayed SIV rebound in RMs and in some cases almost neutralized viral DNA copy detection in serum. Very promising indeed, however there are some questions I wish the authors explored to answer, detailed below.

    Overall, this is an elegant and timely work demonstrating the feasibility of reducing virus rebound in animals, and potentially expand to clinical studies. The work was well written, and sections were clearly discussed.

    Strengths:

    The work is well designed, rationale explained and written very clearly for lay readers.
    Claims are adequately supported by evidence and well designed experiments including controls.

    Weaknesses:

    (1) It looks like ICP0 is also involved in latency reversal effects. More follow-up work will be required to test if this is in fact true.

    (2) It is difficult to estimate the depletion of the latent viral reservoir. The authors have tried to address this issue. A more convincing argument to this reviewer will be data to demonstrate that after the bi-functional vaccine, the animals show overall reduction in the number of circulating latent cells. The feasibility to obtain such a result is not clearly demonstrated.

    (3) The authors state that the reduced virus rebound detected following bi-functional vaccine delivery is due to latent genomes becoming activated and steady-state neutralization of these viruses by antibody response. This needs to be demonstrated. Perhaps cell-culture experiments from specimen taken from animals might help address this issue. In lab cultures one could create environments without antibody responses, under these conditions one would expect higher level of viral loads being released in response to the vaccine in question.

  13. eLife

    Author response:

    The following is the authors’ response to the original reviews.

    Reviewer #1 (Public Review):

    Summary:

    The authors constructed a novel HSV-based therapeutic vaccine to cure SIV in a primate model. The novel HSV vector is deleted for ICP34.5. Evidence is given that this protein blocks HIV reactivation by interference with the NF-kB pathway. The deleted construct supposedly would reactivate SIV from latency. The SIV genes carried by the vector ought to elicit a strong immune response. Together the HSV vector would elicit a shock and kill effect. This is tested in a primate model.

    Thank you for your kind comments and suggestions, which are very helpful in improving our manuscript. We have carefully revised our manuscript and performed additional experiments accordingly, and we now think this version has been substantially improved for your reconsideration.

    Strengths and weaknesses:

    (1) Deleting ICP34.5 from the HSV construct has a very strong effect on HIV reactivation. Why is no eGFP readout given in Figure 1C as for WT HSV? The mechanism underlying increased activation by deleting ICP34.5 is only partially explored. Overexpression of ICP34.5 has a much smaller effect (reduction in reactivation) than deletion of ICP34.5 (strong activation); so the story seems incomplete.

    Thank you for your careful review and kind reminder.

    (1) We are sorry for the misunderstanding of Figure 1C. In the experiment of Figue 1C, we used an HSV-1 17 strain containing GFP (HSV-GFP) and HSV-DICP34.5 (recombinant HSV-1 17 strain with ICP34.5 deletion based on HSV-GFP) to reactivate the HIV latency cell line (J-Lat 10.6 cell). Since detecting GFP cannot distinguish between HSV infection and HIV reactivation, we assessed the reactivation by measuring the mRNA levels of HIV LTR upon stimulation with either HSV-GFP or HSV-ΔICP34.5. Actually, in Figure 1B, we had verified the reactivation efficacy by infecting J-Lat 10.6 cells with the HSV-1 17 strain containing GFP (HSV-GFP) and found significant upregulation of mRNA levels of HIV-1 LTR, Tat, Gag, Vif, and Vpr. We have adjusted the corresponding descriptions accordingly in the revised manuscript.

    (2) We agree with your insightful mention that the mechanism underlying increased activation by HSV-ΔICP34.5 is worthy to be further explored in the future study. In this study, we found that ICP34.5 play an antagonistic role with the reactivation of HIV latency by HSV-1 mainly through the modulation of host NF-κB and HSF1 pathways, while HSV-1 (especially HSV-ΔICP34.5) might reactivate HIV latency through NF-κB, HSF1, and other yet-to-be-determined mechanisms. Thus, ICP34.5 overexpression can only a partial effect on the reduction of the HIV latency reactivation by HSV-1. We have mentioned this issue in the revised “Discussion section”. “Intriguingly, these findings collectively indicated that ICP34.5 might play an antagonistic role in the reactivation of HIV by HSV-1, and thus our modified HSV-DICP34.5 constructs can effectively reactivate HIV/SIV latency through the release of imprisonment from ICP34.5. However, ICP34.5 overexpression had only a partial effect on the reduction of the HIV latency reactivation, indicating that HSV-DICP34.5-based constructs can also reactivate HIV latency through other yet-to-be-determined mechanisms.” (Lines 334 to 340).

    (2) No toxicity data are given for deleting ICP34.5. How specific is the effect for HIV reactivation? An RNA seq analysis is required to show the effect on cellular genes.

    Thank you for your questions and suggestions.

    (1) It’s well known that ICP34.5 is a neurotoxicity factor that can antagonize host immune responses, and previous studies (in gene therapy and oncolytic virotherapy) have shown that the safety of recombinant HSV-based vector can be improved by deleting ICP34.5. In this study, we also found that HSV-DICP34.5 exhibited lower virulence and replication ability than its parental strain (HSV-GFP) (Figure 1D, Figure S1). In addition, HSV-DICP34.5 induced a lower level of inflammatory cytokines (including IL-6, IL-1β, and TNF-α) in primary CD4+ T cells from PLWH compared to HSV-GFP stimulation, likely due to its lower virulence and replication ability (Figure 1I-K). In addition, the CD4+ /CD8+ T cell ratio (Figure 5I) and body weight (Figure S9) after treatment were effectively ameliorated in the SIV-infected macaques of the ART+HSV-DICP34.5-sPD1-SIVgag/SIVenv group. Our data also demonstrated that there was no significant effect on the cell composition of peripheral blood in the SIV-infected macaques of ART+HSV-sPD1-SIVgag/SIVenv group (Figure S10). Thus, these data suggest the safety of HSV-DICP34.5 in PLWH might be tolerable. We have added the corresponding description in the revised manuscript.

    (2) In our study, we found both adenovirus and vaccinia virus cannot reactivate HIV latency (Figure S3). In addition, the deletion of ICP0 gene from HSV-1 diminished the reactivation effect of HIV latency by HSV-1 (Figure S4). Thus, these data suggested the reactivation of HIV latency by HSV-1 might be virus-specific. Of course, this might be further investigated in future studies. We have added the corresponding description in the revised manuscript.

    (3) To explore the mechanism of reactivating viral latency by HSV-DICP34.5-based constructs, we performed RNA-seq analysis (Figure S5). We have added the corresponding description accordingly in the revised manuscript.

    (3) The primate groups are too small and the results to variable to make averages. In Figure 5, the group with ART and saline has two slow rebounders. It is not correct to average those with a single quick rebounder. Here the interpretation is NOT supported by the data.

    We agree with you that this is a pilot study with limited numbers of rhesus macaques. Although the number of macaques was relatively limited, these nine macaques were distributed evenly based on the background level of age, sex, weight, CD4 count, and viral load (VL) (Table S2). All SIV-infected macaques used in this study had a long history of SIV infection and had several courses of ART therapy, which mimics treatment of chronic HIV-1 infection in humans. These macaques were infected with SIVmac239 for more than 5 years, and highly pathogenic SIV-infected macaques have been well-validated as a stringent model to recapitulate HIV-1 pathogenesis and persistence during ART therapy in humans. Indeed, in our Chinese rhesus model, ART treatment effectively suppressed SIV infection to undetectable levels in plasma, and upon ART discontinuation, virus rapidly rebounded, which is very similar with that in ART-treated HIV patients. We think the results of this pilot study were very promising for further studies which will be expanded the scale of animals and then to preclinical and clinical study in our next projects. Thank you for your understanding.

    As for your question regarding “the two animals with low VL and slow rebound”, our explanation is following: As mentioned above, these macaques were distributed evenly based on the background level of CD4 count and VL (Table S2), and then there were different change of viral load and viral rebound in different groups. Thus, we think these data can support our interpretation. Moreover, our conclusion can also be supported from at least three evidences.

    (1) The VL in the ART+saline group promptly rebounded after ART discontinuation, with an average 8.63-fold increase in the rebounded peak VL compared with the pre-ART VL (Figure 5A, D and E). However, plasma VL in the ART+HSV-sPD1-SIVgag/SIVenv group exhibited a delayed rebound interval (Figure 5B-D).

    (2) There was a lower rebounded peak VL than pre-ART VL in the ART+HSV-sPD1-SIVgag/SIVenv group (average 12.20-fold decrease), while a higher rebounded peak VL than pre-ART VL in the ART+HSV-empty group (average 2.74-fold increase) (Figure 5E).

    (3） We found significant suppression of total SIV DNA and integrated SIV DNA provirus in the ART+HSV-sPD1-SIVgag/SIVenv group. However, the copies of the SIV DNA provirus were significantly improved in the ART+HSV-empty group and ART+saline group (Figure 5F-G).

    Thank you for your understanding.

    Discussion

    HSV vectors are mainly used in cancer treatment partially due to induced inflammation. Whether these are suitable to cure PLWH without major symptoms is a bit questionable to me and should at least be argued for.

    Thank you for your kind question comment and question. We confirmed the enhanced reactivation of HIV latency by HSV-∆ICP34.5 in primary CD4+ T cells from people living with HIV (PLWH) (Figure S2). As mentioned above, previous studies have shown that the safety of recombinant HSV-based vector can be improved by deleting ICP34.5. In this study, we also found that HSV-DICP34.5 exhibited lower virulence and replication ability than its parental strain (HSV-GFP) (Figure 1D, Figure S1). In addition, HSV-DICP34.5 induced a lower level of inflammatory cytokines (including IL-6, IL-1β, and TNF-α) in primary CD4+ T cells from PLWH compared to HSV-GFP stimulation, likely due to its lower virulence and replication ability (Figure 1I-K). In addition, the CD4+ /CD8+ T cell ratio (Figure 5I) and body weight (Figure S9) after treatment were effectively ameliorated in the SIV-infected macaques of the ART+HSV-DICP34.5-sPD1-SIVgag/SIVenv group. Our data also demonstrated that there was no significant effect on the cell composition of peripheral blood in the SIV-infected macaques of ART+HSV-sPD1-SIVgag/SIVenv group (Figure S10). Thus, these data suggest the safety of HSV-DICP34.5 in PLWH might be tolerable. We have added the corresponding description in the revised manuscript.

    Reviewer #2 (Public Review):

    Summary:

    In this article, Wen et. al. describe the development of a 'proof-of-concept' bi-functional vector based on HSV-deltaICP-34.5's ability to purge latent HIV-1 and SIV genomes from cells. They show that co-infection of latent J-lat T-cell lines with an HSV-deltaICP-34.5 vector can reactivate HIV-1 from a latent state. Over- or stable expression of ICP 34.5 ORF in these cells can arrest latent HIV-1 genomes from transcription, even in the presence of latency reversal agents. ICP34.5 can co-IP with- and de-phosphorylate IKKa/b to block its interaction with NF-k/B transcription factor. Additionally, ICP34.5 can interact with HSF1 which was identified by mass-spec. Thus, the authors propose that the latency reversal effect of HSV-deltaICP-34.5 in co-infected JLat cells is due to modulatory effects on the IKKa/b-NF-kB and PP1-HSF-1 pathway.

    Next, the authors cleverly construct a bifunctional HSV-based vector with deleted ICP34.5 and 47 ORFs to purge latency and avoid immunological refluxes, and additionally, expand the application of this construct as a vaccine by introducing SIV genes. They use this 'vaccine' in mouse models and show the expected SIV-immune responses. Experiments in rhesus macaques (RM), further elicit the potential for their approach to reactivate SIV genomes and at the same time block their replication by antibodies. What was interesting in the SIV experiments is that the dual-functional vector vaccine containing sPD1- and SIV Gag/Env ORFs effectively delayed SIV rebound in RMs and in some cases almost neutralized viral DNA copy detection in serum. Very promising indeed, however, there are some questions I wish the authors had explored to get answers to, detailed below.

    Overall, this is an elegant and timely work demonstrating the feasibility of reducing virus rebound in animals, with the potential to expand to clinical studies. The work was well-written, and sections were clearly discussed.

    Strengths:

    The work is well designed, rationale explained, and written very clearly for lay readers.
    Claims are adequately supported by evidence and well-designed experiments including controls.

    Thank you for your nice comments regarding our work.

    Weaknesses:

    (1) While the mechanism of ICP34.5 interaction and modulation of the NF-kB and HSF1 pathways are shown, this only proves ICP34.5 interactions but does not give away the mechanism of how the HSV-deltaICP-34.5 vector purges HIV-1 latency. What other components of the vector are required for latency reversal? Perhaps serial deletion experiments of the other ORFs in the HSV-deltaICP-34.5 vector might be revealing.

    Thank you for your valuable suggestion. In fact, we are currently further exploring some potential viral genes of HSV-1 that might play a role in the reactivation of HIV latency. We have found that the deletion of ICP0 gene from HSV-1 diminished the reactivation effect of HIV latency by HSV-1 (Figure S4), showing that ICP0 might play a vital role for the reactivation. Of course, this might be further investigated in future studies. We have added the corresponding description in the revised manuscript.

    (2) The efficacy of the HSV vaccine vectors was evaluated in Rhesus Macaque model animals. Animals were chronically infected with SIV (a parent of HIV), treated with ART, challenged with bi-functional HSV vaccine or controls, and discontinued treatment, and the resulting virus burden and immune responses were monitored. The animals showed SIV Gag and Env-specific immune responses, and delayed virus rebound (however rebound is still there), and below-detection viral DNA copies. What would make a more convincing argument to this reviewer will be data to demonstrate that after the bi-functional vaccine, the animals show overall reduction in the number of circulating latent cells. The feasibility of obtaining such a result is not clearly demonstrated.

    Thank you for your valuable mention. We have now provided more data about this issue. We found significant suppression of total SIV DNA and integrated SIV DNA provirus in the ART+HSV-sPD1-SIVgag/SIVenv group. However, the copies of the SIV DNA provirus were significantly improved in the ART+HSV-empty group and ART+saline group (Figure 5F-G). We have added the corresponding description in the revised manuscript.

    (3) The authors state that the reduced virus rebound detected following bi-functional vaccine delivery is due to latent genomes becoming activated and steady-state neutralization of these viruses by antibody response. This needs to be demonstrated. Perhaps cell-culture experiments from specimens taken from animals might help address this issue. In lab cultures one could create environments without antibody responses, under these conditions one would expect a higher level of viral loads to be released in response to the vaccine in question.

    Thanks for your kind mention and suggestion. We performed the following cell experiment to address this issue. Primary CD4+ T cells from people living with HIV (PLWH) were isolated, and then infected with HSV or HSV-∆ICP34.5 constructs. As expected, we confirmed the enhanced reactivation of HIV latency by HSV-∆ICP34.5 (Figure S2). Thank you.

    (4) How do the authors imagine neutralizing HIV-1 envelope epitopes by a similar strategy? A discussion of this point may also help.

    Thank you for your kind comment. We have added the corresponding discussion in the revised manuscript. “The current consensus on HIV/AIDS vaccines emphasizes the importance of simultaneously inducing broadly neutralizing antibodies and cellular immune responses. Therefore, we believe that incorporating the induction of broadly neutralizing antibodies into our future optimizing approaches may lead to better therapeutic outcomes.” (Lines 384 to 388)

    (5) I thought the empty HSV-vector control also elicited somewhat delayed kinetics in virus rebound and neutralization, can the authors comment on why this is the case?

    Thank you for your careful review and mention. We agree with you that the HSV-1 empty vector does exhibit somewhat a delayed rebound. We think the possible reason is: Although the empty HSV-vector cannot elicit SIV-specific CTL responses, it effectively activates the latent SIV reserviors, and then these activated virions can be partially killed by ART drugs. Therefore, even without carrying HIV/SIV antigens, somewhat delayed kinetics in virus rebound may be observed. Thank you.

    Reviewer #1 (Recommendations For The Authors):

    (1) The authors should provide toxicity data for HSV transduction after deleting ICP34.5 and provide an explanation of why overexpression of ICP34.5 has such a small effect.

    Thank you for your questions and suggestions. As mentioned above, we now provided data for the safety of HSV-DICP34.5-based constructs.

    (1) It’s well known that ICP34.5 is a neurotoxicity factor that can antagonize host immune responses, and previous studies (in gene therapy and oncolytic virotherapy) have shown that the safety of recombinant HSV-based vector can be improved by deleting ICP34.5. In this study, we also found that HSV-DICP34.5 exhibited lower virulence and replication ability than its parental strain (HSV-GFP) (Figure 1D, Figure S1). In addition, HSV-DICP34.5 induced a lower level of inflammatory cytokines (including IL-6, IL-1β, and TNF-α) in primary CD4+ T cells from PLWH compared to HSV-GFP stimulation, likely due to its lower virulence and replication ability (Figure 1I-K). In addition, the CD4+ /CD8+ T cell ratio (Figure 5I) and body weight (Figure S9) after treatment were effectively ameliorated in the SIV-infected macaques of the ART+HSV-DICP34.5-sPD1-SIVgag/SIVenv group. Our data also demonstrated that there was no significant effect on the cell composition of peripheral blood in the SIV-infected macaques of ART+HSV-sPD1-SIVgag/SIVenv group (Figure S10). Thus, these data suggest the safety of HSV-DICP34.5 in PLWH might be tolerable. We have added the corresponding description in the revised manuscript.

    (2) We agree with your insightful mention that the mechanism underlying increased activation by HSV-ΔICP34.5 is worthy to be further explored in the future study. In this study, we found that ICP34.5 play an antagonistic role with the reactivation of HIV latency by HSV-1 mainly through the modulation of host NF-κB and HSF1 pathways, while HSV-1 (especially HSV-ΔICP34.5) might reactivate HIV latency through NF-κB, HSF1, and other yet-to-be-determined mechanisms. Thus, ICP34.5 overexpression can only a partial effect on the reduction of the HIV latency reactivation by HSV-1. We have mentioned this issue in the revised “Discussion section”. “Intriguingly, these findings collectively indicated that ICP34.5 might play an antagonistic role in the reactivation of HIV by HSV-1, and thus our modified HSV-DICP34.5 constructs can effectively reactivate HIV/SIV latency through the release of imprisonment from ICP34.5. However, ICP34.5 overexpression had only a partial effect on the reduction of the HIV latency reactivation, indicating that HSV-DICP34.5-based constructs can also reactivate HIV latency through other yet-to-be-determined mechanisms.” (Lines 334 to 340).

    (2) How specific is the effect for HIV reactivation? An RNA seq analysis is required to show the effect on cellular genes.

    Thank you for your questions and suggestions.

    (1) In our study, we found both adenovirus and vaccinia virus cannot reactivate HIV latency (Figure S3). In addition, the deletion of ICP0 gene from HSV-1 diminished the reactivation effect of HIV latency by HSV-1 (Figure S4). Thus, these data suggested the reactivation of HIV latency by HSV-1 might be virus-specific. Of course, this might be further investigated in future studies. We have added the corresponding description in the revised manuscript.

    (2) To explore the mechanism of reactivating viral latency by HSV-DICP34.5-based constructs, we performed RNA-seq analysis (Figure S5). Results showed that there were numerous differentially expressed genes (DEGs) in response to HSV-ΔICP34.5 infection. Among them, 2288 genes were upregulated, and 611 genes were downregulated. GO analysis showed the enrichment of these DEGs in cellular cycle, cellular development, and cellular proliferation, and KEGG enrichment analysis indicated the enrichment in pathways such as cellular cycle and cytokine-cytokine receptor interaction. We have added the corresponding description accordingly in the revised manuscript.

    (3) A comparison in primates has to be given for constructs with or without ICP34.5 to validate cell culture data (what is an empty vector?)

    Thank you for your reminder. In the revised manuscript, we performed the following cell experiment to address this issue. Primary CD4+ T cells from people living with HIV (PLWH) were isolated, and then infected with HSV or HSV-∆ICP34.5 constructs. As expected, we confirmed the enhanced reactivation of HIV latency by HSV-∆ICP34.5 (Figure S2). Thank you.

    (4) Legends should be improved in writing and content.

    Thank you for your kind mention. In the revised version, we have improved both the manuscript content and the legends of all Figures have been carefully revised in writing and content. Thank you.

    (5) The primate groups should be enlarged before any reliable conclusions can be made. Inflammatory/tox data should be provided.

    Thank you for your question.

    (1) As mentioned above, we agree with you that this is a pilot study with limited numbers of rhesus macaques. Although the number of macaques was relatively limited, these nine macaques were distributed evenly based on the background level of age, sex, weight, CD4 count, and viral load (VL) (Table S2). All SIV-infected macaques used in this study had a long history of SIV infection and had several courses of ART therapy, which mimics treatment of chronic HIV-1 infection in humans. These macaques were infected with SIVmac239 for more than 5 years, and highly pathogenic SIV-infected macaques have been well-validated as a stringent model to recapitulate HIV-1 pathogenesis and persistence during ART therapy in humans. Indeed, in our Chinese rhesus model, ART treatment effectively suppressed SIV infection to undetectable levels in plasma, and upon ART discontinuation, virus rapidly rebounded, which is very similar with that in ART-treated HIV patients. We think the results of this pilot study were very promising for further studies which will be expanded the scale of animals and then to preclinical and clinical study in our next projects. Thank you for your understanding.

    (2) As well known, ICP34.5 is a neurotoxicity factor that can antagonize host immune responses, and previous studies have shown that the safety of recombinant HSV-based vector can be improved by deleting ICP34.5. In this study, we also found that HSV-DICP34.5 exhibited lower virulence and replication ability than its parental strain (HSV-GFP) (Figure 1D, Figure S1). In addition, HSV-DICP34.5 induced a lower level of inflammatory cytokines (including IL-6, IL-1β, and TNF-α) in primary CD4+ T cells from PLWH compared to HSV-GFP stimulation, likely due to its lower virulence and replication ability (Figure 1I-K). In addition, the CD4+ /CD8+ T cell ratio (Figure 5I) and body weight (Figure S9) after treatment were effectively ameliorated in the SIV-infected macaques of the ART+HSV-DICP34.5-sPD1-SIVgag/SIVenv group. Our data also demonstrated that there was no significant effect on the cell composition of peripheral blood in the SIV-infected macaques of ART+HSV-sPD1-SIVgag/SIVenv group (Figure S10). Thus, these data suggest the safety of HSV-DICP34.5 in PLWH might be tolerable. We have added the corresponding description in the revised manuscript.

    (6) Discuss the potential of inflammatory HSV vaccines to be used in PLWH without clinical symptoms.

    Thank you for your mention. As discussed above, we found that HSV-DICP34.5 exhibited lower virulence and replication ability than its parental strain (Figure 1D, Figure S1), and we also found that HSV-DICP34.5 induced a lower level of inflammatory cytokines (including IL-6, IL-1β, and TNF-α) in primary CD4+ T cells from PLWH compared to HSV-GFP stimulation, likely due to its lower virulence and replication ability (Figure 1I-K). In addition, the CD4+ /CD8+ T cell ratio (Figure 5I) and body weight (Figure S9) after treatment were effectively ameliorated in the SIV-infected macaques of the ART+HSV-DICP34.5-sPD1-SIVgag/SIVenv group. Our data also demonstrated that there was no significant effect on the cell composition of peripheral blood in the SIV-infected macaques of ART+HSV-sPD1-SIVgag/SIVenv group (Figure S10). Thus, these data suggest the safety of HSV-DICP34.5 in PLWH might be tolerable. We have added the corresponding description in the revised manuscript.

    Reviewer #2 (Recommendations For The Authors):

    I think the authors have done due diligence to the experimental system, and collected evidence to show the feasibility of delaying virus rebound in macaques. However, I would encourage the authors to perform experiments that can back up the claim that delayed virus rebound is due to neutralization effects, or perhaps due to a reduction in viral reservoir. I believe insights into this process will add rigor, and push the relevance of the study to the next level.

    Thank you for your nice comment and valuable suggestion. We have now provided more data about this issue. We found significant suppression of total SIV DNA and integrated SIV DNA provirus in the ART+HSV-sPD1-SIVgag/SIVenv group. However, the copies of the SIV DNA provirus were significantly improved in the ART+HSV-empty group and ART+saline group (Figure 5F-G). We also discussed that incorporating the induction of broadly neutralizing antibodies into our future optimizing approaches may lead to better therapeutic outcomes in the revised Discussion section. We have added the corresponding description in the revised manuscript. Thank you.

    Altogether, all of the above comments and suggestions are very helpful in improving our manuscript. We have taken these comments into account seriously and try our best to address these questions point-by-point. After making extensive revisions, we now submit this revised manuscript for your re-consideration. Thank you again for all of your comments and suggestions.

  14. Version published to 10.1101/2024.02.20.581122v2 on bioRxiv
  15. Version published to 10.7554/elife.95964.1 on eLife
  16. eLife

    Author response:

    Reviewer #1 (Public Review):

    (1) Deleting ICP34.5 from the HSV construct has a very strong effect on HIV reactivation. Why is no eGFP readout given in Figure 1C as for WT HSV? The mechanism underlying increased activation by deleting ICP34.5 is only partially explored. Overexpression of ICP34.5 has a much smaller effect (reduction in reactivation) than deletion of ICP34.5 (strong activation); so the story seems incomplete.

    Thank you for your comment. (1) In Figure 1c, "HSV-wt" refers to the virus rescued from pBAC—GFP-HSV (as mentioned in the “Method” section), which carries GFP itself. Therefore, detecting GFP cannot distinguish between HSV infection and HIV reactivation. Hence, we assess the reactivation effect by measuring the mRNA levels of HIV LTR. (2) Our data indicate that overexpression of ICP34.5 inhibits the reactivation of the HIV latent reservoir, but this effect is not equivalent to the activation observed in HSV-1 with ICP34.5 deletion. There are some possible reasons: one is that the overexpression of ICP34.5 by lentivirus is randomly integrated into the genome of J-Lat cell line, which will potentially activate HIV latency to some extent. The other is that ICP34.5 mainly inhibited HIV reactivation through modulation of host NF-κB or HSF1 pathways, while PMA, TNF-a, and HSV-1 with deleted ICP34.5 can reactivate HIV latency by other mechanisms that have yet to be determined. Thereby, exerting a synergistic small inhibitory effect. We will further discuss this issue in the revised version. Thank you.

    (2) No toxicity data are given for deleting ICP34.5. How specific is the effect for HIV reactivation? An RNA seq analysis is required to show the effect on cellular genes.

    Thank you for your comment. We plan to conduct several experiments to demonstrate a reduction in HSV-1 replication after ICP34.5 deletion: (1) Detect the growth curve of HSV-1 deleted with ICP34.5 in Vero cells. The virus growth curve of HSV-1 with deleted ICP34.5 may be lower than that of wild-type HSV-1, which could demonstrate a reduction in HSV-1 replication after ICP34.5 deletion. (2) Detect the level of inflammatory factors in tumor cells after infection with HSV-1 deleted with ICP34.5.

    We believe that the effect is specific, as we previously tested poxviruses and adenoviruses and found no activation of the latent reservoir. We consider the activation observed with HSV-1 virus and HSV-1 with deleted ICP34.5 to be specific. We will supplement relevant data in the revised version.

    In addition, we will provide the corresponding RNA-seq data to assess its effect on cellular genes.

    (3) The primate groups are too small and the results to variable to make averages. In Figure 5, the group with ART and saline has two slow rebounders. It is not correct to average those with a single quick rebounder. Here the interpretation is NOT supported by the data.

    We agree with you that this is a pilot study of limited numbers of rhesus macaques. There were only 3 monkeys per group in this study, but our results were encouraging. Although the number of macaques was relatively limited, these nine macaques were distributed very carefully based on age, sex, weight and genotype. All SIV-infected macaques used in this study had a long history of SIV infection and had several courses of ART therapy, which mimics treatment of chronic HIV-1 infection in humans. These macaques were infected with SIVmac239 for more than 5 years, and highly pathogenic SIV-infected macaques have been well-validated as a stringent model to recapitulate HIV-1 pathogenesis and persistence during ART therapy in humans. Indeed, in our rhesus model, ART treatment effectively suppressed SIV infection to undetectable levels in plasma, and upon ART discontinuation, virus rapidly rebounded, which is very similar with that in ART-treated HIV patients. Our further studies will be expanded the scale of animals and then to preclinical and clinical study in our next projects. Thank you for your understanding.

    Discussion

    HSV vectors are mainly used in cancer treatment partially due to induced inflammation. Whether these are suitable to cure PLWH without major symptoms is a bit questionable to me and should at least be argued for.

    We will provide more data about the safety assessment of HSV-1 vector in SIV-infected macaques, and also further discuss the potential of inflammatory HSV vector in PLWH in the revised manuscript.

    Reviewer #2 (Public Review):

    (1) While the mechanism of ICP34.5 interaction and modulation of the NF-kB and HSF1 pathways are shown, this only proves ICP34.5 interactions but does not give away the mechanism of how the HSV-deltaICP-34.5 vector purges HIV-1 latency. What other components of the vector are required for latency reversal? Perhaps serial deletion experiments of the other ORFs in the HSV-deltaICP-34.5 vector might be revealing.

    We agree with your suggestion. In fact, we are currently further exploring some viral genes of HSV-1 that play a role in activation. We have found that the ICP0 gene of HSV-1 virus can activate HIV, and the specific mechanism is under investigation.

    (2) The efficacy of the HSV vaccine vectors was evaluated in Rhesus Macaque model animals. Animals were chronically infected with SIV (a parent of HIV), treated with ART, challenged with bi-functional HSV vaccine or controls, and discontinued treatment, and the resulting virus burden and immune responses were monitored. The animals showed SIV Gag and Env-specific immune responses, and delayed virus rebound (however rebound is still there), and below-detection viral DNA copies. What would make a more convincing argument to this reviewer will be data to demonstrate that after the bi-functional vaccine, the animals show overall reduction in the number of circulating latent cells. The feasibility of obtaining such a result is not clearly demonstrated.

    Thank you for your suggestion. We will plan to conduct IPDA experiments to further supplement data on the overall reduction in circulating latent cell numbers in animals.

    (3) The authors state that the reduced virus rebound detected following bi-functional vaccine delivery is due to latent genomes becoming activated and steady-state neutralization of these viruses by antibody response. This needs to be demonstrated. Perhaps cell-culture experiments from specimens taken from animals might help address this issue. In lab cultures one could create environments without antibody responses, under these conditions one would expect a higher level of viral loads to be released in response to the vaccine in question.

    We plan to use primary cells for related experiments to further validate the results of the cell experiments.

    (4) How do the authors imagine neutralizing HIV-1 envelope epitopes by a similar strategy? A discussion of this point may also help.

    Thank you for your comments. In fact, our study adopts the "shock and kill" strategy, with a focus on the "kill" aspect leaning towards T-cell therapy. Although the vaccine in the paper also utilizes Env antigen, we believe these antibodies are insufficient for neutralizing the mutated SIV virus. We strongly agree with your suggestion that in HIV/AIDS treatment, effective T-cell killing combined with broad-spectrum neutralizing antibodies would be more effective. This aligns with our findings, as our treatment has partially delayed viral rebound but with a relatively short duration of suppression. This may indicate insufficient killing activity. In future research, we will further consider the role of broad-spectrum neutralizing antibodies. Our revised manuscript will elaborate on this in the discussion section.

    (5) I thought the empty HSV-vector control also elicited somewhat delayed kinetics in virus rebound and neutralization, can the authors comment on why this is the case?

    We agree with you that the HSV-1 empty vector does exhibit somewhat a delayed rebound. The reason is that our treatment simultaneously utilizes both the HSV vector vaccine and ART therapy. Although the empty HSV-vector cannot elicit SIV-specific CTL response, it effectively activates the latent SIV reservoirs and then these activated virions can be partially killed by ART, Therefore, even without carrying antigens, the slight delay may be achieved.

  17. eLife

    eLife assessment

    In this useful study, the authors tested a novel approach to eradicate the HIV reservoir by constructing a herpes simplex virus (HSV)-based therapeutic vaccine. The approach was tested in experimental infections of chronically SIV-infected, antiretroviral therapy (ART)-treated macaques with extent of rebound after ART interruption as a measure of the size of the HIV reservoir. While mean viremia at rebound was lower in the HSV vaccine-treated group, the evidence presented appear to be be incomplete because the group size was small and the viral load at rebound was highly variable.

  18. eLife

    Reviewer #1 (Public Review):

    Summary:

    The authors constructed a novel HSV-based therapeutic vaccine to cure SIV in a primate model. The novel HSV vector is deleted for ICP34.5. Evidence is given that this protein blocks HIV reactivation by interference with the NFkappaB pathway. The deleted construct supposedly would reactivate SIV from latency. The SIV genes carried by the vector ought to elicit a strong immune response. Together the HSV vector would elicit a shock and kill effect. This is tested in a primate model.

    Strengths and weaknesses:

    (1) Deleting ICP34.5 from the HSV construct has a very strong effect on HIV reactivation. Why is no eGFP readout given in Figure 1C as for WT HSV? The mechanism underlying increased activation by deleting ICP34.5 is only partially explored. Overexpression of ICP34.5 has a much smaller effect (reduction in reactivation) than deletion of ICP34.5 (strong activation); so the story seems incomplete.

    (2) No toxicity data are given for deleting ICP34.5. How specific is the effect for HIV reactivation? An RNA seq analysis is required to show the effect on cellular genes.

    (3) The primate groups are too small and the results to variable to make averages. In Figure 5, the group with ART and saline has two slow rebounders. It is not correct to average those with a single quick rebounder. Here the interpretation is NOT supported by the data.

    Discussion

    HSV vectors are mainly used in cancer treatment partially due to induced inflammation. Whether these are suitable to cure PLWH without major symptoms is a bit questionable to me and should at least be argued for.

  19. eLife

    Reviewer #2 (Public Review):

    Summary:

    In this article, Wen et. al. describe the development of a 'proof-of-concept' bi-functional vector based on HSV-deltaICP-34.5's ability to purge latent HIV-1 and SIV genomes from cells. They show that co-infection of latent J-lat T-cell lines with an HSV-deltaICP-34.5 vector can reactivate HIV-1 from a latent state. Over- or stable expression of ICP 34.5 ORF in these cells can arrest latent HIV-1 genomes from transcription, even in the presence of latency reversal agents. ICP34.5 can co-IP with- and de-phosphorylate IKKa/b to block its interaction with NF-k/B transcription factor. Additionally, ICP34.5 can interact with HSF1 which was identified by mass-spec. Thus, the authors propose that the latency reversal effect of HSV-deltaICP-34.5 in co-infected JLat cells is due to modulatory effects on the IKKa/b-NF-kB and PP1-HSF-1 pathway.

    Next, the authors cleverly construct a bifunctional HSV-based vector with deleted ICP34.5 and 47 ORFs to purge latency and avoid immunological refluxes, and additionally, expand the application of this construct as a vaccine by introducing SIV genes. They use this 'vaccine' in mouse models and show the expected SIV-immune responses. Experiments in rhesus macaques (RM), further elicit the potential for their approach to reactivate SIV genomes and at the same time block their replication by antibodies. What was interesting in the SIV experiments is that the dual-functional vector vaccine containing sPD1- and SIV Gag/Env ORFs effectively delayed SIV rebound in RMs and in some cases almost neutralized viral DNA copy detection in serum. Very promising indeed, however, there are some questions I wish the authors had explored to get answers to, detailed below.

    Overall, this is an elegant and timely work demonstrating the feasibility of reducing virus rebound in animals, with the potential to expand to clinical studies. The work was well-written, and sections were clearly discussed.

    Strengths:

    The work is well designed, rationale explained, and written very clearly for lay readers.

    Claims are adequately supported by evidence and well-designed experiments including controls.

    Weaknesses:

    (1) While the mechanism of ICP34.5 interaction and modulation of the NF-kB and HSF1 pathways are shown, this only proves ICP34.5 interactions but does not give away the mechanism of how the HSV-deltaICP-34.5 vector purges HIV-1 latency. What other components of the vector are required for latency reversal? Perhaps serial deletion experiments of the other ORFs in the HSV-deltaICP-34.5 vector might be revealing.

    (2) The efficacy of the HSV vaccine vectors was evaluated in Rhesus Macaque model animals. Animals were chronically infected with SIV (a parent of HIV), treated with ART, challenged with bi-functional HSV vaccine or controls, and discontinued treatment, and the resulting virus burden and immune responses were monitored. The animals showed SIV Gag and Env-specific immune responses, and delayed virus rebound (however rebound is still there), and below-detection viral DNA copies. What would make a more convincing argument to this reviewer will be data to demonstrate that after the bi-functional vaccine, the animals show overall reduction in the number of circulating latent cells. The feasibility of obtaining such a result is not clearly demonstrated.

    (3) The authors state that the reduced virus rebound detected following bi-functional vaccine delivery is due to latent genomes becoming activated and steady-state neutralization of these viruses by antibody response. This needs to be demonstrated. Perhaps cell-culture experiments from specimens taken from animals might help address this issue. In lab cultures one could create environments without antibody responses, under these conditions one would expect a higher level of viral loads to be released in response to the vaccine in question.

    (4) How do the authors imagine neutralizing HIV-1 envelope epitopes by a similar strategy? A discussion of this point may also help.

    (5) I thought the empty HSV-vector control also elicited somewhat delayed kinetics in virus rebound and neutralization, can the authors comment on why this is the case?

  20. Version published to 10.1101/2024.02.20.581122v1 on bioRxiv