Systems vaccinology aims to improve understanding of vaccine outcomes, by using unbiased approaches to identify the major associated immune phenotypes, including peripheral blood cell population frequencies and gene expression (Pulendran et al., 2010). This has been particularly informative for dissecting the molecular and cellular correlates of natural human variation that can influence vaccine responses and outcomes in vivo (Tsang, 2015). Sex and prior exposure to the same or similar antigen are prominent examples of natural variation that influence vaccinee responses. However, immune response differences associated with sex and prior exposure have typically been defined and analyzed separately. These variables have not been examined together because vaccination studies often involve individuals who are either almost entirely previously exposed (e.g., influenza studies Bucasas et al., 2011; Nakaya et al., 2016; Nakaya et al., 2011; Tsang et al., 2014; Kotliarov et al., 2020) or naive (e.g., Ebola or yellow fever studies in US cohorts Gaucher et al., 2008; Querec et al., 2009; Rechtien et al., 2017) to the vaccine pathogen. Here, we utilize a unique cohort of herpes simplex virus (HSV) 2 vaccine recipients that includes both sexes, as well as naive and previously exposed subjects, thus allowing us to study the in vivo molecular and cellular response correlates of the joint effects of sex and prior exposure.

Sex is a well-known variable that can influence vaccine and disease responses. Females typically develop higher antibody responses and report more adverse reactions following vaccination than males (Klein et al., 2015). These differences could be a result of multiple sex dimorphic traits, including sex hormones and increased expression of X-linked genes escaping inactivation. Females in particular have been observed to show higher levels of Toll-like receptor (TLR)/interferon (IFN)-associated gene expression in innate responses to vaccination (Klein et al., 2010). Systems immunology studies have identified prevaccination differences that predict antibody titer responses, such as CD20⁺CD38^high B cell frequency and plasmacytoid dendritic cell activation (Kotliarov et al., 2020). Prior exposure to antigen is another dominant factor in immune response, but its effects have most often been analyzed focusing on adaptive, antigen-specific responses. An example of particular interest for innate responses involved vaccine vectors using replication-defective viruses: trials testing an experimental adenovirus vector-based HIV vaccine revealed increased inflammatory responses in subjects seropositive for a prevalent adenovirus subtype and showed an enhanced risk of HIV infection that was associated with prior adenovirus exposure (Zak et al., 2012; Buchbinder et al., 2008; Gray et al., 2011).

Here, we analyzed a unique cohort receiving a multidose, replication-defective vaccine, to interrogate the interaction of sex and prior exposure to infection. Subjects received an experimental HSV2 vaccine in a phase 1 clinical trial where comparisons could be made between three groups of volunteers based on their HSV serostatus prior to vaccination: HSV1⁻/HSV2⁻, HSV1⁺/HSV2⁻, or HSV1^±/HSV2⁺ (Dropulic et al., 2019). Each of these groups included subjects from both sexes. HSV2 is an important human pathogen. Persons infected with HSV2 have a threefold increased risk of acquiring HIV, and HSV2 infection causes genital herpes and severe disease in neonates, patients with AIDS, and transplant recipients (Koelle and Corey, 2008; Freeman et al., 2006). Prior attempts to produce an effective prophylactic vaccine for HSV2, including glycoprotein subunit or replication-competent vaccines, have been unsuccessful (Dropulic and Cohen, 2012). The replication-defective vaccine HSV529 studied here is deleted for two essential HSV genes, UL5 and UL29, and propagated in a cell line expressing these two proteins (Da Costa et al., 1999; Da Costa et al., 2000). Infection of cells not expressing these two essential genes results in expression of nearly all the viral genes, but viral DNA replication is abolished, and no virions are produced. The vaccine induced greater than fourfold increases in HSV-specific antibody titers in most HSV1⁻/HSV2⁻ vaccine recipients, but lower increases in antibody responses were observed in volunteers in the other serogroups; T cell responses were modest in all three serogroups (Dropulic et al., 2019).

We characterized immune responses longitudinally by analyzing blood transcriptomic profiles and high-dimensional immune cell phenotyping throughout the three-dose vaccine regimen (Dropulic et al., 2019). Vaccine-induced changes and correlates of outcomes such as neutralizing antibody titers and adverse reactions to the vaccine were determined. The transcriptomic responses were associated with both sex and prior exposure to HSV, but the responding genes and pathways also differed markedly based on the combination of both variables. Women showed stronger early inflammatory and type I IFN responses when compared to men, but this was only observed for HSV seronegative vaccine recipients. Thus, using unbiased systems immunology analyses, we show how prior exposure and sex interact to shape early, innate immune responses to a replication-defective vaccine, and identify transcriptional profiles that may shape the subsequent immune responses induced in vivo.

To understand the interaction between sex and prior exposure in the context of an immune response in vivo, we studied the response to a replication-defective HSV2 vaccine in persons with different prior exposure to HSV. We used a systems immunology approach characterizing peripheral blood transcriptomic and cell population phenotypes. While prior studies using systems approaches have been reported for live attenuated virus, subunit, and vectored vaccines, this is the first such study of a replication-defective vaccine in humans. After a longitudinal analysis throughout the three-dose vaccine regimen, we focused on transcriptional responses at day 1 after the first dose, as these exhibited the most robust changes associated with both sex and prior exposure. Changes in these phenotypes were determined and comparisons were made with other outcomes, such as the observation of more robust vaccination responses in women than men in subjects previously naive to HSV, revealed by faster neutralizing antibody responses. Thus, we provide insights for how prior exposure and sex interact to shape the early innate immune response.

The day 1 transcriptional responses differed between HSV529 vaccine recipients based on prior exposure to HSV. Responses to HSV529 by HSV seronegative individuals overlapped strongly with those previously reported for recipients of the yellow fever vaccine, whereas responses to HSV529 by HSV seropositive individuals were significantly different from those who were HSV seronegative and instead overlapped with persons who had been vaccinated with influenza. These findings support the notion that prior exposure to the pathogen against which the vaccine is targeting is a major determinant of the early innate immune response to the vaccine, independent of the vaccine type (live attenuated, subunit, or replication-defective) or the pathogen itself (yellow fever virus, influenza virus, or HSV).Interestingly, IFN responses appeared to be skewed toward IFN-α in the HSV seronegative vaccine recipients but toward IFN-γ in the HSV seropositive group. Type I IFNs, including IFN-α, are produced very early after viral infections, including with HSV, and cellular RNA and DNA sensors for HSV produce type I IFNs in response to virus infection (Oh et al., 2016; Danastas et al., 2020). IFN-γ signals through a different receptor and is predominantly produced by natural killer cells and T cells during infection (Platanias, 2005). IFN-γ is particularly important for inhibiting HSV reactivation and thus may be more prominent when the immune system has previously been exposed to the virus (Decman et al., 2005). Both IFNs contribute to the induction of an antiviral state through the upregulation of molecules that can antagonize virus replication, but are increasingly being recognized to exert regulatory effects on the innate response that influence development of adaptive immunity (Lee and Ashkar, 2018).

Sex was associated with day 1 transcriptional responses, but the enriched pathways differed between HSV naive and seropositive individuals. A transcriptional profile of increased responses was observed for women, specifically for those previously naive to HSV. The transcriptomic modules and leading-edge genes that characterized this response were centered on type I IFN and antiviral pathways. This suggests that in the absence of prior exposure, the more robust transcriptional responses in women to a replication-defective virus may be due to an underlying difference in baseline inflammation or in the speed of induction of inflammation in comparison to men. Such IFN-associated broad immune activation has recently been found to underlie shared set point signatures for increased vaccine responsiveness in healthy individuals of both sexes (although this signature tends to be more elevated in females than males at baseline), as well as disease activity in patients with lupus (Kotliarov et al., 2020). Thus, the more robust day 1 transcriptional responses in HSV naive women could have contributed to the more rapid neutralizing antibody responses observed in HSV naive women in this cohort. Interestingly, we found that within HSV naive women the transcriptional IFN response correlated negatively with vaccine-induced antibody titers. Possibly, as HSV529 is a replication-defective vaccine dependent on an initial infection of host cells, early IFN responses inhibited the number of cells infected or the efficiency of viral antigen expression. This is consistent with the established role of IFN antagonizing natural HSV infection (Leib, 2002) and indicates how the effects of IFN responses on vaccine effectiveness may be context dependent. Further work will be necessary toassess whether similar observations can be replicated in other naive responses to replication-defective or live attenuated vaccines, including similar consequences for the induction of antibody titers.

Despite the IFN response on day 1 that was associated with reduced antibody titers in HSV naive women, these individuals as a group showed the most robust antibody responses to HSV529, with more rapid antibody responses compared to HSV naive men. Overall, HSV naive individuals showed greater titer increases compared to HSV exposed subjects particularly at later timepoints. The diminished responses by HSV exposed subjects may also have resulted from suppression of HSV529 infection, in this case by preexisting neutralizing antibody or T cell immunity. This is supported by the large number of genes upregulated on day 1 after vaccination in HSV1⁻/HSV2⁻ persons in comparison with the much smaller number of genes upregulated in these same individuals with subsequent doses of vaccine, or in HSV1⁺/HSV2⁻ or HSV1^±/HSV2⁺ subjects at any timepoint. Evidence for preexisting antibody inhibiting live virus vaccination has been reported for other pathogens such as measles, for which efficacy improves if vaccination is administered later within the first year of life; clinical data combined with experimental models indicate this is more likely due to interference from maternal antibodies rather than immaturity of the neonatal system (Aaby et al., 2014; van Binnendijk et al., 1997), although the underlying mechanisms remain largely unclear. Another example is the live attenuated intranasal influenza vaccine which is approved in the United States for persons aged 2–49; prior exposure to influenza may lead to neutralization and reduced replication of the live influenza virus vaccine resulting in reduced efficacy in adults compared to children (Hoft et al., 2017; Monto et al., 2009; Grohskopf et al., 2021). In HSV exposed subjects a higher dose of HSV529 may be required to achieve a better response; indeed the live attenuated zoster vaccine is given at 14 times the dose of the varicella vaccine, in part, to overcome the effect of preexisting immunity (Cohen, 2008).

More robust responses by women compared to men have been described for several vaccines (Klein et al., 2015), and our findings demonstrate some overlap with prior studies of sex differences in the context of HSV. In two double-blind randomized studies of an HSV subunit vaccine, the vaccine was protective against genital herpes in women who were HSV1⁻/HSV2⁻ prior to vaccination, but the vaccine was not effective in men (Stanberry et al., 2002). A subsequent large double-blind placebo-controlled trial found that women reported more symptoms and developed higher levels of antibody than did men after vaccination; however, the differences in antibody titers were not significant, and a later large study focused on HSV1⁻/HSV2⁻ women failed to replicate efficacy against HSV2 infection or disease (Bernstein et al., 2005; Belshe et al., 2012). Murine studies have also implicated IFN responses in association with sex in the context of HSV. Male mice have a higher mortality rate after HSV infection than females which correlates with the difference in IFN-γ response, and male IFN-γ knockout mice have higher HSV reactivation rates than control mice whereas female IFN-γ knockout mice show no difference from controls (Han et al., 2001). We now show that in the context of human responses to HSV529, sex-associated differences in IFN and inflammatory signaling are significantly more pronounced in women not previously exposed to HSV.

Prior studies suggest that HSV2 subunit vaccines are more effective in women than men (Stanberry et al., 2002), but the underlying mechanisms of the sex-dependent response may be different for the live, replication-defective HSV2 vaccine used in our study. For example, HSV is known to activate TLR9, and thus a replication-defective vaccine like ours would be expected to activate TLR9 by presenting viral DNA to the immune system. TLR9 responses are known to be sex dependent, with a role in autoimmune disorders disproportionately affecting females, and may have thus contributed to the more robust innate responses in females in our study (Lund et al., 2003; Marshak-Rothstein, 2006). A replication-defective live viral vaccine may also be more effective in triggering the presentation of processed viral peptides on the surface of the infected host cells to stimulate innate and virus-specific T cell responses compared to protein subunit vaccines. Thus, our HSV2 replication-defective vaccine may be more likely to trigger innate and cellular immune response pathways that are more robust in women, for example, similar to earlier observations of more robust IFN-α production and CD8 T cell activation after stimulation of PBMCs with a viral peptide in HIV-infected women compared to men (Meier et al., 2009).

Due to the more rapid vaccine response in seronegative women observed in this study, we analyzed changes in plasmablasts. In a trial of the malaria recombinant protein vaccine RTS,S, increased plasmablasts were observed on day 1, but only after the second and third doses of vaccine (Kazmin et al., 2017). We detected expansion of plasmablasts when analyzing all subjects at both days 1 and 7 after the first dose of vaccine, but these changes did not reach statistical significance when stratifying by prior exposure and sex. We also observed that the numbers of plasmablasts peaked around day 7 as others have reported, although the early response we observed at day 1 was unexpected (Wrammert et al., 2008; Magnani et al., 2017).

Several individual genes were highlighted by our transcriptomic screen. TLR7 is an X-linked gene for which higher levels of expression have been reported in B cells of naive female versus male mice after vaccination with inactivated influenza vaccine, and these higher levels of TLR7 correlated with high levels of antibodies in the female mice (Fink et al., 2018). We also detected higher levels of TLR7 gene expression in HSV1⁻/HSV2⁻ women compared to men on day 1 after vaccination. TLR7 is important for immunoglobulin class switching and could represent a molecular mechanism for sex-based differences, which have been attributed to X-linked immune response genes as well as to hormonal differences (Harper and Flanagan, 2018; Klein and Flanagan, 2016). A further observation arising from the broad phenotypic characterization is the concordance between significant host gene expression responses and pathogen immune evasion. The most significantly upregulated individual genes were dominated at day 1 by IFN-stimulated (CXCL10, GBP1, and IFIT2) or Fcγ receptor genes (FCGR1A, FCGR1B, and FCGR1C). At day 7, the most significantly upregulated individual genes included integrins (ITGB3, ITGB5, and ITGA2B). HSV encodes immune evasion proteins that antagonize many of these specific gene functions: blocking IFN, antibody-mediated cytolysis, and activity of integrin β3 (Leib, 2002; Lubinski et al., 1998; Gianni et al., 2012).

Overall, our study confirms and adds to the substantial effects observed for prior exposure and sex in innate immune responses. We document at a systems level how these two variables interact in the context of response to a replication-defective vaccine, and determine resulting transcriptional response profiles that may form the basis for differences in neutralizing antibody responses in vivo. To improve vaccination effectiveness, it will thus be important to better understand how prior exposure and sex affect the innate responses that are increasingly being shown to shape subsequent adaptive immunity.

Microarray data, cell population frequencies, and neutralizing antibody titers are available at GEO (accession number GSE185341). Transcriptome data for 9 of the 60 vaccine recipients obtained at days 180 and 187 after immunization is excluded from the GEO database since blood was drawn on these participants at those timepoints after genomic data sharing language was revised and this new language was not added to the participant's consent forms.

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Decision letter after peer review:

Thank you for submitting your article "Sex and prior exposure jointly shape innate immune responses to a replication-defective herpesvirus vaccine" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Arturo Casadevall as the Senior Editor. The following individuals involved in the review of your submission have agreed to reveal their identity: Assya Trofimov (Reviewer #1); Sita Awasthi (Reviewer #3).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

1. If data is available, the authors should check the enrichment of TLR7 (or lack thereof) in the placebo group to rule out the potential vaccine-driven effects.

2. Extend the discussion on the BTM analysis in the Methods section. More details are necessary.

3. Discuss the possibility of sex-specific variations in immunological outcomes after vaccination with a replication-defective virus compared to an adjuvanted subunit vaccine.

4. Modify line 162 (or Figure S1) so the message is consistent.

5. Please follow reviewers' suggestions to improve the readability of figures throughout the manuscript.

6. Please correct the typos listed by the reviewers.

Reviewer #1 (Recommendations for the authors):

Sex and prior exposure to the antigen are known factors that influence the immune response to vaccines. In this study, the authors study how the combination of these factors changes the immune response to a multi-dose replication-defective herpes simplex virus (HSV) vaccine.

Strengths

The main strength of this study is the variability in the data that was acquired and the granularity of the annotation. The authors meticulously established comparisons between the vaccine recipient subgroups and came up with interesting results and conclusions.

Weakness

Some analyses were challenging to understand and the methods section would have benefited from additional details.

I would have wanted to see a more robust conclusion about the loss of sex-specific differences in antigen-experienced individuals compared to naive individuals. This conclusion is alluded to but not explicitly made.

The authors achieved their aims in quantifying the difference in the immune response between naive individuals and those with previous exposure to HSV as well as differences between males and females. Their results fully support the conclusions stated in the manuscript.

This manuscript provides a comprehensive assessment of the differences between male and female responders as well as naive and virus-experienced responders. These results show many interesting leads for a better understanding of these differences. These results are especially important in the context of the development of vaccines against viruses that establish lifelong infections, such as HSV, CMV and VZV. Since the authors have committed to releasing the data, this will likely offer other teams some rich data for subsequent analysis.

I. Comments and questions:

a) Analysis for Figures B and C: Since all of the Δ_Day0 Neutralizing Ab Titers are at zero on Day 0, I'm not sure the statistical test is accurate. The test compares means and since all values have a mean of zero on the first day (post normalization) and necessarily do not have a mean of zero at the next time points (as per the normalization procedure), this makes me conclude that the test is likely biased. Perhaps changing the strategy would be best. The authors seem to want to verify if the difference between men and women is significant. Perhaps simply comparing the values at the second timepoint would be a better use of a statistical test and reflect reality. So my suggestion is:

ttest(a, b) where a = women's normalized titer @ 30 Days and b = men's normalized titer @ 30 Days.

b) Line 162: This statement is not entirely true. Since Supplementary Figure 1 only shows recorded responses for HSV- individuals (as per legend description), only based on that figure it is impossible to state that prior exposure markedly affects the response to the vaccine. These results are not even in that Figure! If they are, the legend should be adjusted, if not, they should be added to match the conclusion!

c) Figure 2, 3, 4: For each of these Figures, the manuscript would benefit from clear indications of which subgroups are being compared.

d) Methods should contain a clear description of the BTM analysis: how was it done? Which software was used? What are the gene sets used?

e) I am curious about Dose 2. What happened to it? Why is there such a low cell enrichment (Figure 2A) and such a low number of differentially expressed genes (Figure 2B)? Was the immune response sustained from Day 7 to Dose 3 and this is why there are few differentially expressed genes? Was there a problem in the quantification?

f) Why did the authors combine all responders for Figure 2C when they suspected differences in sex and between naive/antigen-experienced groups?

g) I really like the analysis done in Figure 2D. I think the link to other vaccine studies is interesting. Since DEGs are often very dependent on the dataset, perhaps repeating the comparison but with enriched pathways or even the BTMs would be more convincing. I feel like the signal is slightly blurred with the DEGs and Jaccard index since DEGs are often dataset-specific and the Jaccard index is so highly dependent on the overall set size. I suspect the authors will find a greater signal using pathways instead.

h) Line 246: IL-12 has some influence on Th1 adaptive immune response. Perhaps splitting the results from Figure 2C into seronegative and seropositive would have been better and would show the difference in IL-12-based Th1 cell enrichment.

i) Line 300: The authors are correct to note that TLR7 is located on chromosome X. Would it be possible to check within the placebo group comparing males and females if TLR7 is still significantly enriched? This would rule out any vaccine-based effect compared to the sex-based only effect.

j) Figure 4 and Lines 334 and 336. Authors state similar pathway enrichments but then conclude that there is a difference in the effect of sex between seropositive and seronegative groups. In Figure 4B, it seems like the biggest difference is seen between seronegative individuals. And yet there are the same genes enriched in.

k) Figure 4A. Perhaps some additional explanation could reconcile this contradiction in this paragraph!

Line 370: So if I understand correctly, seropositive females lose the high IFN response because of a higher neutralizing titer (as seen in seronegative/positive males?)

l) Data was not available and is currently embargoed until October 2024!

Reviewer #2 (Recommendations for the authors):

The authors study responses to a non-replicating human HSV vaccine. They find that the vaccine responses differ in males and females, predominantly in those individuals who are HSV seronegative.

Strengths.

The authors have a relatively large cohort of patients enabling longitudinal studies in seronegative and seropositive male and female individuals

The authors performed a comprehensive study of the immune responses both on the level of neutralizing antibodies and gene activation for long time periods

Their work identified clear differences between males and females in seronegative individuals: females produce neutralizing ab's faster, but males have delayed but higher levels.

The work is able to relate some of the differences to the differences in the Type I IFN response.

Weaknesses.

Many of the observations reported in the paper have already been described in the literature, such as sex-specific immune responses to viral vaccines.

The possible causal connection between the kinetics of gene activation and the differences in ab responses is discussed but requires further work.

The paper leaves uncertain if this vaccine is going to be epidemiologically/clinically useful (as these are Phase 1 clinical trials).

I am uncertain what is the significance of these results for understanding the fundamental biology of the immune responses to vaccination.

I think this is a technically very strong paper that reports interesting results

However, I am not sure it reports a breakthrough in our understanding of either the vaccine responses or the sex-linked differences.

I think this is important work that might be more appropriate in a human immunology/vaccinology journal.

Reviewer #3 (Recommendations for the authors):

This study describes early antiviral innate responses of a replication-defective herpesvirus vaccine and the subsequent impact on adaptive responses based on the sex of the vaccinee and prior exposure to the virus. As early as one day after the vaccination the differences depended on sex and prior exposure to the virus were noted using blood transcriptomics and cell population profiling. Type I interferon (IFN) signature was found to be the most pronounced in HSV naive women and associated with lower neutralizing antibody titers, suggesting that a strong early antiviral response reduced the uptake of this replication-defective virus vaccine.

Strengths:

The methodology used by the authors has a wider implication beyond the replication-defective herpes virus vaccine.

This is the first detailed study using the systems immunology approach to assess early immune responses and transcriptomic analysis for a replication-defective herpesvirus vaccine.

The authors have provided a robust data set to justify the conclusions.

Figures are clear and statistical analysis is vigorous.

Weaknesses:

Overall, this manuscript has minimal weaknesses.

The manuscript shows that neutralizing activity is negatively correlated with IFN type 1 signatures in females, it would be of great interest to analyze whether overall gD2 binding or HSV-2 binding antibody levels are also lower in females compared to males or only the antibody with neutralizing activity.

If total binding antibody levels are not different, analyzing the Fc receptor-mediated activity such as ADCC would be of great interest.

A few recommendations are made to improve the manuscript

1. Since this study most likely used the same sets of samples that were used in a prior study (Dropulic et al. JID 2019 published by the same group), it may be possible to discuss whether the difference in binding antibody was noted. Alternatively, the data may be further analyzed by sex for discussion purposes.

2. Day 1 following 1st immunization seems to show the most interesting pattern in HSV naïve population. Conceptually, with a replication-defective viral vaccine, the subsequent immunizations in the HSV naive group are more likely to behave similar to that of the virus-exposed group and consequently, immune responses may be indicative of that. It is not clear whether later time point samples were assessed and analyzed based on the naive and prior exposure group.

3. Volcano plot for figure 2B clearly shows the changes in individual genes in samples collected over time. The Y-axis scale variations among panels minimize the appearance of huge differences. If it is difficult to maintain the same scale for all the panels, a comment can be inserted in the figure legend to note the differences in the scale.

4. Previously published phase III trials with subunit vaccines showed higher efficacy in the HSV naïve women. It is to be noted that higher efficacy in females was not assessed for correlation with neutralizing activity in the prior study. One of the most interesting findings presented here is that a replication-defective vaccine had a smaller boost in neutralizing activity in females following 2nd immunization compared to men. It may be interesting to discuss the possibility of variations in immunological outcome based on the sex with a replication-defective viral vaccine compared to an adjuvanted subunit vaccine.

Essential revisions:

1. If data is available, the authors should check the enrichment of TLR7 (or lack thereof) in the placebo group to rule out the potential vaccine-driven effects.

TLR7 expression is profiled, including for recipients of placebo stratified by gender, in Figure 3—figure supplement 2. This shows that the TLR7 response is not observed in placebo recipients of either gender, and this is now highlighted in the text (line 325).

2. Extend the discussion on the BTM analysis in the Methods section. More details are necessary.

TLR7 expression is profiled, including for recipients of placebo stratified by gender, in Figure 3—figure supplement 2. This shows that the TLR7 response is not observed in placebo recipients of either gender, and this is now highlighted in the text (line 325).

3. Discuss the possibility of sex-specific variations in immunological outcomes after vaccination with a replication-defective virus compared to an adjuvanted subunit vaccine.

We now discuss in a dedicated paragraph in the discussion (lines 466-479), potential mechanisms underlying differences in responses between sexes in the context of a replication-defective versus a subunit protein vaccine. Among those are that TLR9 and CD8 T cell pathways may be driven more strongly by the replication-defective vaccine, and that these have previously been reported to show sex-specific variation.

4. Modify line 162 (or Figure S1) so the message is consistent.

Figure 1—figure supplement 1 has now been modified to include HSV+ in addition to HSV- individuals, and we have modified the text, now lines 220-225. These continue to support that adverse reactions and titer responses are associated with both sex and prior exposure.

5. Please follow reviewers' suggestions to improve the readability of figures throughout the manuscript.

Corrections have been made, such as to the axis of Figures 4C and D, Figure 3—figure supplement 2, and to the legends of Figures2,3,4.

6. Please correct the typos listed by the reviewers.

Corrections have been made such as to the parenthesis on line 55 and text on line 95

Reviewer #1 (Recommendations for the authors):

Sex and prior exposure to the antigen are known factors that influence the immune response to vaccines. In this study, the authors study how the combination of these factors changes the immune response to a multi-dose replication-defective herpes simplex virus (HSV) vaccine.

Strengths

The main strength of this study is the variability in the data that was acquired and the granularity of the annotation. The authors meticulously established comparisons between the vaccine recipient subgroups and came up with interesting results and conclusions.

Weakness

Some analyses were challenging to understand and the methods section would have benefited from additional details.

See response to essential revisions comments 2 and 5 above.

I would have wanted to see a more robust conclusion about the loss of sex-specific differences in antigen-experienced individuals compared to naive individuals. This conclusion is alluded to but not explicitly made.

See response to essential revision comment 3 above

The authors achieved their aims in quantifying the difference in the immune response between naive individuals and those with previous exposure to HSV as well as differences between males and females. Their results fully support the conclusions stated in the manuscript.

This manuscript provides a comprehensive assessment of the differences between male and female responders as well as naive and virus-experienced responders. These results show many interesting leads for a better understanding of these differences. These results are especially important in the context of the development of vaccines against viruses that establish lifelong infections, such as HSV, CMV and VZV. Since the authors have committed to releasing the data, this will likely offer other teams some rich data for subsequent analysis.

I. Comments and questions:

a) Analysis for Figures B and C: Since all of the Δ_Day0 Neutralizing Ab Titers are at zero on Day 0, I'm not sure the statistical test is accurate. The test compares means and since all values have a mean of zero on the first day (post normalization) and necessarily do not have a mean of zero at the next time points (as per the normalization procedure), this makes me conclude that the test is likely biased. Perhaps changing the strategy would be best. The authors seem to want to verify if the difference between men and women is significant. Perhaps simply comparing the values at the second timepoint would be a better use of a statistical test and reflect reality. So my suggestion is:

ttest(a, b) where a = women's normalized titer @ 30 Days and b = men's normalized titer @ 30 Days.

We feel that performing a δ using the baseline at day 0, i.e. (D30-D0) and then analyzing this using Mann-Whitney-Wilcoxon for either males or females separately within the seronegative group is an informative approach to assess whether a more robust titer responses after vaccination are displayed by seronegative females. This showed a significant response in the female group (p<0.001) but insignificant change for males, consistent with the observation that increases in titer at day 30 are seen for 7 out 10 females but only 1 out of the 5 males.

We also performed the test suggested by this reviewer. This specific test yields p=0.1, even though our analysis described above did highlight the possibility of responses distinct to seronegative females. We thus think that we are likely not powered to assess significant differences in titer responses between groups. However, as shown in the manuscript, we subsequently investigated further by analyzing immune phenotypes and showed, at the transcriptional level, significantly different responses in seronegative females compared to males or seropositive subjects (Figures3 and 4). These data together support our overall conclusion.

b) Line 162: This statement is not entirely true. Since Supplementary Figure 1 only shows recorded responses for HSV- individuals (as per legend description), only based on that figure it is impossible to state that prior exposure markedly affects the response to the vaccine. These results are not even in that Figure! If they are, the legend should be adjusted, if not, they should be added to match the conclusion!

We have revised the text, now lines 220-225, as well as included responses for HSV+ individuals in the figure, now Figure 1—figure supplement 1.

c) Figure 2, 3, 4: For each of these Figures, the manuscript would benefit from clear indications of which subgroups are being compared.

We have revised the figure legends to be clear which subgroups are being compared. The legend to Figure 2A states in the first sentence that all groups are being compared (no subgroups) and we have now stated this for the legend for Figure 2B and 2C. Figure 2D indicates we used subgroups based on HSV serology. The first sentence of the legend to figure 3 states that the figure is analyzing HSV seronegative vaccine recipients based on sex. The first sentence of the legend to figure 4 states that the figure is analyzing vaccine recipients based on HSV serology and sex (the bottom of panel A shows the subgroups and the top of panels B and C show the subgroups), while the legend for panel D indicates it shows HV seronegative women.

d) Methods should contain a clear description of the BTM analysis: how was it done? Which software was used? What are the gene sets used?

See response to essential revision comment 2 above.

e) I am curious about Dose 2. What happened to it? Why is there such a low cell enrichment (Figure 2A) and such a low number of differentially expressed genes (Figure 2B)? Was the immune response sustained from Day 7 to Dose 3 and this is why there are few differentially expressed genes? Was there a problem in the quantification?

The immune responses appeared much higher to the first dose of vaccine than to either the 2^nd (day 30) or 3^rd (day 180) doses. Note the large difference in scale of the y axis in figure 2B day7, 37, and 187 compared to day1, and in response to the reviewers comment we now highlight this in the legend for figure 2B. There were not technical problems when quantifying response to dose 2, although we did not collect blood on days 31 and 188 (day 1 after the 2^nd and 3^rd doses of the vaccine) and may have missed the peak of those responses. There was also not sustained transcriptomic perturbation at the day of the second does, indicated by very few DEG comparing between day 30 and day 0. Differential responses between doses have been documented for other vaccines, and may reflect altered responsiveness particularly when re-vaccinating within a short period of 30 days (dose 2) compared to 150 days (dose 3).

f) Why did the authors combine all responders for Figure 2C when they suspected differences in sex and between naive/antigen-experienced groups?

Despite the potential sex/exposure effects, in Figure 2A-C we initially combined all subjects to first characterize what the vaccine is doing to the immune system in terms of transcriptomic and cell population responses overall. We then go on to explore effects of sex and prior exposure in subsequent figures.

g) I really like the analysis done in Figure 2D. I think the link to other vaccine studies is interesting. Since DEGs are often very dependent on the dataset, perhaps repeating the comparison but with enriched pathways or even the BTMs would be more convincing. I feel like the signal is slightly blurred with the DEGs and Jaccard index since DEGs are often dataset-specific and the Jaccard index is so highly dependent on the overall set size. I suspect the authors will find a greater signal using pathways instead.

We appreciate this suggestion but, given the analysis using DEGs already showed a clear effect, we have not gone on to explore the analysis at the pathway level to avoid inundating the reader with additional data (since this is already an omics level study with a dense presentation of data). We agree it could show a greater signal but feel the important point of overlap with other vaccines based on prior exposure is already shown, and that the particular pathways involved are also well characterized in those prior studies of vaccines in which all recipients are either naïve (yellow fever) or previously exposed (influenza).

h) Line 246: IL-12 has some influence on Th1 adaptive immune response. Perhaps splitting the results from Figure 2C into seronegative and seropositive would have been better and would show the difference in IL-12-based Th1 cell enrichment.

Our intention for Figure 2A-C was to initially combine all subjects to first characterize what the vaccine is doing to the immune system. Subsequently we focused on transcriptomic rather than flow cytometry responses to assess effects of prior exposure to HSV. Although we appreciate the point about IL-12 and Th1 populations specifically, overall we feel that the transcriptomic responses represent an unbiased approach and have more power based on the greater response observed. Therefore, we have remained focused on the transcriptomic rather than flow data to investigate the effects of prior exposure.

i) Line 300: The authors are correct to note that TLR7 is located on chromosome X. Would it be possible to check within the placebo group comparing males and females if TLR7 is still significantly enriched? This would rule out any vaccine-based effect compared to the sex-based only effect.

See response to essential revisions comment 1.

j) Figure 4 and Lines 334 and 336. Authors state similar pathway enrichments but then conclude that there is a difference in the effect of sex between seropositive and seronegative groups. In Figure 4B, it seems like the biggest difference is seen between seronegative individuals. And yet there are the same genes enriched in.

The contrasts assessed in Figure 4 demonstrate that effects of sex within either seropositive group are statistically different from the effects of sex within seronegative individuals. The text, now lines 336-339, then highlights that the pathways which differed most in both of these comparisons were also those which differed when effect of sex was compared within seronegative subjects alone (Figure 3). These results confirm that the effect of sex on transcriptional response to HSV529 vaccination, which we first observed to be strongest for seronegative subjects in Figure 3, is statistically significant when comparing between naïve and HSV-exposed individuals.

k) Figure 4A. Perhaps some additional explanation could reconcile this contradiction in this paragraph!

Line 370: So if I understand correctly, seropositive females lose the high IFN response because of a higher neutralizing titer (as seen in seronegative/positive males?)

Our interpretation is that high early IFN response by HSV naïve women may have inhibited the number of cells this live-attenuated vaccine could infect, and this impaired the titer of the subsequent antibody responses. Please see discussion lines 423-425.

l) Data was not available and is currently embargoed until October 2024!

We will make the data available when the paper is accepted.

Reviewer #2 (Recommendations for the authors):

The authors study responses to a non-replicating human HSV vaccine. They find that the vaccine responses differ in males and females, predominantly in those individuals who are HSV seronegative.

Strengths.

The authors have a relatively large cohort of patients enabling longitudinal studies in seronegative and seropositive male and female individuals

The authors performed a comprehensive study of the immune responses both on the level of neutralizing antibodies and gene activation for long time periods

Their work identified clear differences between males and females in seronegative individuals: females produce neutralizing ab's faster, but males have delayed but higher levels.

The work is able to relate some of the differences to the differences in the Type I IFN response.

Weaknesses.

Many of the observations reported in the paper have already been described in the literature, such as sex-specific immune responses to viral vaccines.

Sex-specific immune responses to viral vaccines.

We agree sex effects have been reported. However, the combined effects of sex and prior exposure have not. Here we evaluated three different subject groups to dissect the effects of sex when in subjects with: (1) no prior exposure, (2) exposure to prior HSV1, and (3) exposure to prior HSV2. We found significant differences in the effect of sex between these groups, both in innate and adaptive responses.

The possible causal connection between the kinetics of gene activation and the differences in ab responses is discussed but requires further work.

We agree but we feel that further analysis is beyond the scope of this current work. For example, further work will be necessary to determine if the more robust IFN responses observed here in women can be replicated in other naïve responses to vaccination with replication-defective or live attenuated vaccines, and whether such more robust early responses can lead to similar consequences for differential induction of antibody titers. If so, the possibility that a robust early IFN responses in HSV naïve women can inhibit infection or antigen expression by live vaccines would warrant further investigation.

The paper leaves uncertain if this vaccine is going to be epidemiologically/clinically useful (as these are Phase 1 clinical trials).

The prior clinical study report on this vaccine (Dropulic et al. now reference 16) discusses these points. Briefly, the HSV529 vaccine was safe and elicited modest levels of neutralizing antibody responses in HSV-seronegative vaccinees indicating potential as a prophylactic vaccine, although modifications will likely be required to improve its immunogenicity. This paper focuses on the unique opportunity of a cohort with a controlled immune stimulus of vaccination across subjects stratified by both sex and prior exposure, enabling study of how these variables interact to shape an immune response in vivo.

I am uncertain what is the significance of these results for understanding the fundamental biology of the immune responses to vaccination.

This paper emphasizes the interplay between prior exposure to viruses and sex-dependent responses to vaccination. Immune imprinting due to prior exposure is becoming an increasingly important topic affecting the response to many pathogens and vaccines, and sex-specific differences in immune responses to vaccination and infection and disease are becoming increasingly appreciated as fundamental to vaccinology. Our work point to the importance of studying both together as their effects could be dependent on each other.

I think this is a technically very strong paper that reports interesting results

However, I am not sure it reports a breakthrough in our understanding of either the vaccine responses or the sex-linked differences.

I think this is important work that might be more appropriate in a human immunology/vaccinology journal.

Please see responses to reviewer 2’s earlier comments above.

Reviewer #3 (Recommendations for the authors):

This study describes early antiviral innate responses of a replication-defective herpesvirus vaccine and the subsequent impact on adaptive responses based on the sex of the vaccinee and prior exposure to the virus. As early as one day after the vaccination the differences depended on sex and prior exposure to the virus were noted using blood transcriptomics and cell population profiling. Type I interferon (IFN) signature was found to be the most pronounced in HSV naive women and associated with lower neutralizing antibody titers, suggesting that a strong early antiviral response reduced the uptake of this replication-defective virus vaccine.

Strengths:

The methodology used by the authors has a wider implication beyond the replication-defective herpes virus vaccine.

This is the first detailed study using the systems immunology approach to assess early immune responses and transcriptomic analysis for a replication-defective herpesvirus vaccine.

The authors have provided a robust data set to justify the conclusions.

Figures are clear and statistical analysis is vigorous.

Weaknesses:

Overall, this manuscript has minimal weaknesses.

The manuscript shows that neutralizing activity is negatively correlated with IFN type 1 signatures in females, it would be of great interest to analyze whether overall gD2 binding or HSV-2 binding antibody levels are also lower in females compared to males or only the antibody with neutralizing activity.

If total binding antibody levels are not different, analyzing the Fc receptor-mediated activity such as ADCC would be of great interest.

While we have data on gD2 binding, we focused on neutralizing antibody here as it is thought to be more important as a correlate of protection than simply binding to the soluble gD we have used in our assay. We do not have data on ADCC on most of the subjects in this study. We feel these experiments are beyond the scope of this paper.

A few recommendations are made to improve the manuscript

1. Since this study most likely used the same sets of samples that were used in a prior study (Dropulic et al. JID 2019 published by the same group), it may be possible to discuss whether the difference in binding antibody was noted. Alternatively, the data may be further analyzed by sex for discussion purposes.

While we have data on gD2 binding, we focused on neutralizing antibody here as it is thought to be more important as a correlate of protection than simply binding to the soluble gD we have used in our assay.

2. Day 1 following 1st immunization seems to show the most interesting pattern in HSV naïve population. Conceptually, with a replication-defective viral vaccine, the subsequent immunizations in the HSV naive group are more likely to behave similar to that of the virus-exposed group and consequently, immune responses may be indicative of that. It is not clear whether later time point samples were assessed and analyzed based on the naive and prior exposure group.

While we have data on gD2 binding, we focused on neutralizing antibody here as it is thought to be more important as a correlate of protection than simply binding to the soluble gD we have used in our assay.

3. Volcano plot for figure 2B clearly shows the changes in individual genes in samples collected over time. The Y-axis scale variations among panels minimize the appearance of huge differences. If it is difficult to maintain the same scale for all the panels, a comment can be inserted in the figure legend to note the differences in the scale.

We have added to the figure legend in 2B. “Note that the scale of the y axes different on the different days that were analyses.”

4. Previously published phase III trials with subunit vaccines showed higher efficacy in the HSV naïve women. It is to be noted that higher efficacy in females was not assessed for correlation with neutralizing activity in the prior study. One of the most interesting findings presented here is that a replication-defective vaccine had a smaller boost in neutralizing activity in females following 2nd immunization compared to men. It may be interesting to discuss the possibility of variations in immunological outcome based on the sex with a replication-defective viral vaccine compared to an adjuvanted subunit vaccine.

Please see response to essential revision comment 3 above.

Author details

1. Foo Cheung

   Center for Human Immunology, National Institutes of Health, Bethesda, United States

   Contribution

   Formal analysis, Investigation, Writing – review and editing, Data curation, Methodology, Software, Validation, Visualization

   Contributed equally with

   Richard Apps

   Competing interests

   No competing interests declared

2. Richard Apps

   Center for Human Immunology, National Institutes of Health, Bethesda, United States

   Contribution

   Investigation, Writing – review and editing, Project administration, Resources, Writing – original draft

   Contributed equally with

   Foo Cheung

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-5140-0141

3. Lesia Dropulic

   Medical Virology Section, Laboratory of Infectious Diseases, National Institutes of Health, Bethesda, United States

   Present address

   Vaccine Research Center, National Institutes of Health, Bethesda, United States

   Contribution

   Conceptualization, Investigation, Resources, Writing – review and editing

   Competing interests

   No competing interests declared

4. Yuri Kotliarov

   Center for Human Immunology, National Institutes of Health, Bethesda, United States

   Contribution

   Formal analysis, Methodology, Resources

   Competing interests

   No competing interests declared

5. Jinguo Chen

   Center for Human Immunology, National Institutes of Health, Bethesda, United States

   Contribution

   Investigation, Methodology, Validation

   Competing interests

   No competing interests declared

6. Tristan Jordan

   Medical Virology Section, Laboratory of Infectious Diseases, National Institutes of Health, Bethesda, United States

   Present address

   George Washington University School of Medicine and Health Sciences, Washington, DC, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

7. Marc Langweiler

   Center for Human Immunology, National Institutes of Health, Bethesda, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

8. Julian Candia

   Center for Human Immunology, National Institutes of Health, Bethesda, United States

   Contribution

   Formal analysis, Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-5793-8989

9. Angelique Biancotto

   Center for Human Immunology, National Institutes of Health, Bethesda, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

10. Kyu Lee Han

    Center for Human Immunology, National Institutes of Health, Bethesda, United States

    Contribution

    Investigation

    Competing interests

    No competing interests declared

11. Nicholas Rachmaninoff

    Multiscale Systems Biology Section, Laboratory of Immune System Biology, National Institutes of Health, Bethesda, United States

    Contribution

    Resources

    Competing interests

    No competing interests declared

12. Harlan Pietz

    Medical Virology Section, Laboratory of Infectious Diseases, National Institutes of Health, Bethesda, United States

    Present address

    Weill Cornell Medicine and The Rockefeller University, New York, United States

    Contribution

    Resources

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-4792-5708

13. Kening Wang

    Medical Virology Section, Laboratory of Infectious Diseases, National Institutes of Health, Bethesda, United States

    Contribution

    Investigation, Resources

    Competing interests

    No competing interests declared

14. John S Tsang

    1. Center for Human Immunology, National Institutes of Health, Bethesda, United States
    2. Multiscale Systems Biology Section, Laboratory of Immune System Biology, National Institutes of Health, Bethesda, United States

    Present address

    Department of Immunobiology and Center for Systems and Engineering Immunology, Yale University School of Medicine; Department of Biomedical Engineering, Yale University, New Haven, United States

    Contribution

    Conceptualization, Supervision, Project administration, Writing – review and editing, Funding acquisition, Methodology, Resources, Writing – original draft

    Contributed equally with

    Jeffrey I Cohen

    For correspondence

    john.tsang@yale.edu

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-3186-3047

15. Jeffrey I Cohen

    Medical Virology Section, Laboratory of Infectious Diseases, National Institutes of Health, Bethesda, United States

    Contribution

    Conceptualization, Funding acquisition, Writing – original draft, Project administration, Resources, Supervision, Writing – review and editing

    Contributed equally with

    John S Tsang

    For correspondence

    jcohen@niaid.nih.gov

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-0238-7176

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