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

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.

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?

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.