Association between low levels of HIV-1 DNA and HLA class I molecules in chronic HIV-1 infection.

BACKGROUND: HLA-B27 and -B57 were found in people with low levels of HIV-1 DNA, suggesting that HLA class I molecules may influence the size of HIV-1 reservoir. Aim of the study was to explore the association between HLA class I molecules and HIV-1 DNA in people with chronic HIV-1 infection.
METHODS: Post-hoc analysis of the APACHE trial, on adults with chronic HIV-1 infection, prolonged suppressive antiretroviral therapy and good immunological profile. HIV-1 DNA was quantified in peripheral blood mononuclear cells (PBMCs); HLA-A, -B and -C were tested on genomic DNA. Crude odds ratios (OR) with their respective 95% Wald confidence intervals (95% CIs) were estimated by univariable logistic regression for HLAs with a p-value <0.10.
RESULTS: We found 78 and 18 patients with HIV-1 DNA ≥100 copies/106PBMCs and with HIV-1 DNA <100 copies/106PBMCs, respectively. HLA-A24 was present in 21 (29.6%) participants among subjects with HIV-1 DNA ≥100 copies/106PBMCs and 1 (5.9%) among those with HIV-1 DNA <100 copies/106PBMCs (OR = 5.67, 95%CI = 0.79-46.03; p = 0.105); HLA-B39 was present in 1 (1.4%) with HIV-1 DNA ≥100 copies/106PBMCs and in 3 (17.6%) with HIV-1 DNA <100 copies/106PBMCs (OR = 13.71, 95%CI = 1.33-141.77; p = 0.028) and HLA-B55 in 3 (4.2%) and 3 (17.6%), respectively (OR = 4.43, 95%CI = 0.81-24.29; p = 0.087). All the three patients with HLA-B39 and HIV-1 DNA <100 copies/106PBMCs did not have HLA-A24.
CONCLUSIONS: In patients with HIV-1 infection who maintained a good virological and immunological profile, HLA-B39 and -B55 may be associated with lower levels of HIV-1 DNA.

Introduction

Over the past years, the role of host genetic factors in HIV-1 replication and disease progression to acquired immune deficiency syndrome (AIDS) has been widely investigated [1, 2].

In particular, human leukocyte antigen (HLA) is a group of highly polymorphic cell-surface proteins that play a key role in the regulation of immune system, presenting peptide antigens to cytotoxic T lymphocytes (CTLs) to eliminate the infected cells [3]. However, mechanisms of HIV-1 escape from CTL pressure have been previously reported [4].

Among major histocompatibility complex molecules, HLA class I alleles (-A, -B and -C) have demonstrated to be more significantly involved in the HIV-1 pathogenesis; especially, HLA-B appears to have the strongest influence on HIV outcome [5].

Therefore, several studies have focused attention on HLA class I profile of long term non progressors (LTNPs) to better identify determinants related to the spontaneous control of viral replication; nowadays, it is well documented that HLA-B27 and -B57 are associated with a slower HIV-1 disease progression [6, 7], while HLA-B35 with a poor prognosis [8].

Moreover, HLA-B27 and -B57 were also found in LTNPs and elite controllers with low levels of proviral HIV-1 DNA [9, 10], suggesting that HLA class I molecules may have an impact on the size of latent HIV-1 reservoir. Furthermore, data from four analytic treatment interruption trials have revealed an association between HLA-B alleles, including HLA-B27 and -B57, and a delayed viral rebound [11], emphasizing the role of cellular host immunity in controlling HIV-1 replication. These findings are raising interest since one of the greatest challenges towards a HIV cure is to characterize the viral reservoir.

Aim of the study was to explore if there were HLA class I alleles associated with low HIV-1 DNA in people living with HIV-1 (PLWH) chronic infection, prolonged suppressive antiretroviral therapy (ART) and good immunological profile.

Methods

This is a post-hoc exploratory analysis of the APACHE trial, conducted at Infectious Diseases Clinic of the San Raffaele Hospital, Milan, Italy. The APACHE study is a prospective, open-label, single-arm, non-randomized, proof-of-concept study designed to characterize viral rebound during analytic treatment interruption. Subjects with chronic HIV-1 infection, HIV-1 RNA <50 copies/mL for ≥10 years, absence of plasma residual viremia for ≥5 years without any viral blips and CD4+ >500 cells/μL were screened for HIV-1 DNA [12]; long-term non-progressors and elite controllers were excluded. The study protocol was in accordance with the Declaration of Helsinki.

The Ethical Committe of the San Raffaele Hospital approved the study protocol (on 17/05/2016; approval reference number: 31/2016) and all the enrolled patients provided written informed consent.

The APACHE study is registered with ClinicalTrials.gov (NCT03198325, first posted on 26/06/2017).

HIV-1 RNA was quantified by kinetic polymerase chain reaction (PCR) molecular system (Abbott real-time PCR). Undetectable viral load was defined as HIV-1 RNA <50 copies/mL, residual viremia as detectable values of HIV-1 RNA <50 copies/mL (semi-quantitative result), while absence of residual viremia as the lack of detectable HIV-1 RNA in a sample (qualitative result).

At screening, total HIV-1 DNA was amplified as formerly described and quantified in peripheral blood mononuclear cells (PBMCs) by Real Time PCR (ABI Prism 7900) [13]; low HIV-1 DNA was established as HIV-1 DNA <100 copies/106 PBMCs.

At HIV-1 DNA testing, HLA-A, -B and -C were tested on genomic DNA using a PCR sequence-specific oligonucleotide (HISTO SPOT SSO) and a PCR sequence-specific primer (Olerup SSP kits).

All tests were performed in the Laboratory of Microbiology and Virology of IRCCS San Raffaele Scientific Institute.

Patients’ characteristics were reported as median (quartile1-quartile3) or frequency (%). Baseline characteristics were compared using the chi-square test/Fisher’s exact test or the Wilcoxon rank-sum test.

Crude odds ratios (OR) with their respective 95% Wald confidence intervals (95% CIs) were also estimated by univariable logistic regression for HLAs with a p-value <0.10.

All statistical tests were two-sided at the 5% level and were performed using SAS software (version 9.4; SAS Institute, Cary, NC).

Results

At the time of enrolment, PLWH followed at San Raffaele Hospital were 4921: 99 met all the eligibility criteria of the APACHE study and 96 were screened for HIV-1 DNA. Overall, 78 (81%) had HIV-1 DNA ≥100 copies/106PBMCs and 18 (19%) <100 copies/106PBMCs.

At HIV-1 DNA testing, median age was 32 (25.2–38.9), 61 (64%) were male and 15 (15.6%) had a diagnosis of AIDS; furthermore, ART was started 7.9 (2.1–47.8) months after HIV diagnosis and received for a median time of 18.1 (15.6–19.8) years. HIV-1 RNA was <50 copies/mL for a median time of 11.7 (10.7–14.0) years and absence of plasma residual viremia for 6.9 (6.1–7.2) years. Current CD4+ count was 763 (605–922) cells/μL and CD4+/CD8+ ratio 1.35 (0.86–2.02), while nadir CD4+ T cell count was 253 (167–339) cells/μL among patients with HIV-1 DNA ≥100 copies/106PBMCs and 353 (212–434) cells/μL among those with HIV-1 DNA <100 copies/106PBMCs (p = 0.055). Overall, 66 (68.8%) PLWH had a previous virological failure: in more detail, 57 (73.1%) in people with HIV-1 DNA ≥100 copies/106PBMCs and 9 (50%) in people with HIV-1 DNA <100 copies/106PBMCs (p = 0.088). The most frequent treatment regimen was a non-nucleoside reverse transcriptase inhibitor + 2 nucleoside reverse transcriptase inhibitors in both groups [in 34 (43.6%) and 8 (44.4%), respectively].

Other patients’ characteristics are described in Table 1.

Table 1

Participants’ characteristics at HIV-1 DNA testing.

Characteristic	Category	HIV-1 DNA ≥100 copies/106PBMCs (n = 78)	HIV-1 DNA <100 copies/106PBMCs (n = 18)	p-value§
Age at HIV diagnosis (years)		31.7 (25.2–37.7)	34.0 (27.9–40.3)	0.426a
Male gender		47 (60%)	14 (78%)	0.187b
AIDS diagnosis	No	65 (83.3%)	16 (88.9%)	0.729b
	Yes	13 (16.7%)	2 (11.1%)
HIV risk factor	Heterosexual	39 (50%)	6 (33%)	0.034b
	MSM	18 (23%)	3 (17%)
	PWID	10 (13%)	0
	Other	11 (14%)	9 (50%)
HCV coinfection	No	54 (69.2%)	16 (88.9%)	0.224b
	Yes	21 (27%)	2 (11.1%)
	Unknown	3 (3.8%)	0
Nadir CD4+ (cells/μL)		253 (167–339)	353 (212–434)	0.055a
Zenith HIV-1 RNA (copies/mL)		51000 (11000–137708)	31078 (7200–81919)	0.542a
Year of ART start		1998 (1996–2000)	1999 (1997–2002)	0.194a
Months to ART start		12.0 (1.9–51.5)	6.0 (2.8–24.1)	0.526a
ART duration (years)		18.4 (15.7–20.2)	17.5 (14.5–19.0)	0.233a
Years of HIV-1 RNA <50 copies/mL		11.6 (10.6–13.5)	12.3 (10.9–15.2)	0.210a
Years of HIV-1 RNA <1 copy/mL		6.9 (6.1–7.1)	6.8 (6.4–7.5)	0.649a
HIV-1 DNA (copies/106PBMC)		1100 (230–2475)	-	NA
CD4+ cell count (cells/μL)		826 (575–949)	744 (660–773)	0.884a
CD4+%		41.4 (32.2–50.8)	37.8 (26.7–47.6)	0.602a
CD8+ cell count (cells/μL)		632 (533–772)	523 (377–1230)	0.937a
CD8+%		30.1 (25.8–40.5)	28.6 (23.6–41)	0.692a
CD4+/CD8+ ratio		1.38 (0.93–2.07)	1.35 (0.62–2.02)	0.579a

Results described by use of median (IQR) or frequency (%).

§ p-values by Wilcoxon rank-sum test (a) or chi-square/Fisher’s exact test (b).

Abbreviations: PBMC, peripheral blood mononuclear cells; AIDS, acquired immunodeficiency syndrome; MSM, men who have sex with men; PWID, people who inject drugs; ART, antiretroviral therapy; NA, not applicable.

Among 96 participants screened for HIV-1 DNA, 88 were tested for HLA class I profile: we identified 71 (81%) patients with HIV-1 DNA ≥100 copies/106PBMCs and 17 (19%) with HIV-1 DNA <100 copies/106PBMCs.

At HIV-1 DNA determination, HLA class I molecules more frequently observed in subjects with HIV-1 DNA <100 copies/106PBMCs were HLA-A2, described in 11 (64.7%) participants, HLA-A11, -C3, -C6 and -C12, each represented in 4 (23.5%) participants and HLA-C7 in 8 (47.1%), as shown in Fig 1.

Fig 1

HLA class I (HLA-A, -B and–C) alleles are reported in the Fig 1 according to HIV-1 DNA values. HLA class I alleles more frequently observed in subjects with HIV-1 DNA <100 copies/106PBMCs were HLA-A2, -A11, -C3, -C6, -C7 and -C12.

Overall, only 3 HLA class I molecules showed at least a marginal association with HIV-1 DNA: HLA-A24 was present in 21 (29.6%) participants among subjects with HIV-1 DNA ≥100 copies/106PBMCs and 1 (5.9%) among those with HIV-1 DNA <100 copies/106PBMCs (OR = 5.67, 95%CI = 0.79–46.03; p = 0.105); HLA-B39 in 1 (1.4%) with HIV-1 DNA ≥100 copies/106PBMCs and in 3 (17.6%) with HIV-1 DNA <100 copies/106PBMCs (OR = 13.71, 95%CI = 1.33–141.77; p = 0.028) and HLA-B55 in 3 (4.2%) and 3 (17.6%), respectively (OR = 4.43, 95%CI = 0.81–24.29; p = 0.087).

All the three patients with HLA-B39 and HIV-1 DNA <100 copies/106PBMCs did not have HLA-A24.

Discussion

We have focused our attention on HLA class I molecules, in order to explore if they may be associated with a low peripheral viral reservoir in subjects with HIV-1 chronic infection.

Our findings suggest that in particular HLA-B39 and -B55 might be associated with HIV-1 DNA <100 copies/106PBMCs. HLA-B39 was formerly found to be associated with LTNPs in a Spanish cohort [14] and with a lower viremia among PLWH in Zambia [15], consistently with our findings; therefore, both the studies have confirmed the influence of HLA-B39 on the control of viral replication.

However, HLA-B39 antigen was also previously classified as a risk allele due to a higher frequency described in seropositive compared to seronegative subjects, suggesting a role in the susceptibility to HIV-1 infection [16, 17].

For the first time, our analysis has mentioned HLA-B55, a split antigen from the B22 broad antigen, among the alleles detected in PLWH with a long-lasting virological suppression. The protective effect of HLA-B55 was already investigated in a study conducted in Argentina aiming to evaluate allele prevalence in HIV-1 positive people; this HLA allele was absent in HIV-1 infected patients, but present in control subjects [16]. However, HLA-B55, together with -B56, has also demonstrated to predispose to high levels of viremia and a faster progression to AIDS both in European and North American HIV-1 populations [18, 19]; multicenter studies conducting on a greater number of participants should be designed to clarify the role of HLA-B55 in HIV-1 infection.

Among individuals with HIV-1 DNA ≥100 copies/106PBMCs, we observed a significant association with HLA-A24 that was present in nearly a third of PLWH with a higher viral reservoir.

In line with our data, the Multicenter AIDS Cohort Study showed that HLA-A24 was frequent in rapid CD4+ cells decliners [20], supposing that it might be a factor involved in HIV-1 disease progression; as evidence of the unfavorable impact of HLA-A24 antigen, it is rare in LTNPs [14].

The only association reported in literature between low HIV-1 DNA levels and HLA alleles was seen in LTNPs and elite controllers with HLA-B27 and -B57 [9, 10]. In our analysis, these protective alleles were unexpectedly described with a low frequency among people with HIV-1 DNA <100 copies/106PBMCs and we did not find any correlation with the size of HIV-1 reservoir; further investigations on a larger sample size will be helpful to better interpret these findings.

In addition, discordant results from studies may be caused by different HIV-1 positive subjects’ characteristics; in fact, recent data has revealed the importance of performing genome-wide association study to identify ethnical disparities in the host control of HIV-1 infection [21, 22].

One of the limitations of the study is the retrospective design that cannot exclude the persistence of residual confounding. Moreover, the small number of available patients, due to the restricted inclusion criteria applied in the APACHE study, limited the statistical power of the study and the generalizability of our findings. Nevertheless, the major strength relies on the availability of HIV-1 DNA values and then the opportunity to assess the original association between HLA class I alleles and a low HIV-1 peripheral reservoir.

In conclusion, in people with chronic HIV-1 infection, prolonged suppressive ART, absence of plasma residual viremia for ≥5 years and good immunological profile, the presence of HLA-B39 and -B55 may be associated with a lower level of total HIV-1 DNA, highlighting the need to deeply evaluate the role of HLA class I in affecting the size of reservoir in further larger studies.

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Reviewer #1: 1) The three reported signals are 21/78 vs. 1/18 (p=0.105), 1/78 vs. 3/16 (p=0.028), and 3/78 vs. 3/18 (p=0.087). If consider multiple testing adjustment, none is significant. Overall, there is no statistical power the declare any significant signal for the current study. More data or confirmation studies are needed.

Reviewer #2: This aimed at exploring the correlation between low levels of HIV-1 DNA and specific HLA alleles. Although results should be confirmed in a larger sample size, the study is interesting and clearly reported and discussed. However there are some minor points to be addressed:

- Please, report in more details the methodology used to assess the quantification of HIV-DNA and of residual viremia. This part can be also included in the supplementary material.

- Please, specify if the assay allows to quantify total or integrated HIV-DNA.

- Please, better specify the sentence “HIV-1 RNA was <50 copies/mL for a median time of 11.7 (10.6-14.0) years and absence of plasma residual viremia for 6.9 (6.2-7.2) years”. Does it mean that HIV-1 RNA or residual viremia was persistently undetectable or absent throughout the median time reported, with no episodes of viral blips?

- Please, modify “Nadir CD4+ were” in “Nadir CD4+ T cell count was”

- Which was the selection criteria for the 88 patients screened for HLA?

- In the footnotes of Table 1, please report the statistical tests used to assess statistical significance.

- In Figure 1, please highlight those HLA alleles significantly associated with different levels of HIV-1 DNA.

- Is there a correlation between HLA and the outcome of analytical treatment interruption?

Reviewer #3: I suggest to better clarify some aspects:

- More information about study population should be useful: year of enrolment, type of regimens and line of ARV, if they ever failed… Moreover, since the discussion includes consideration about LTNP and elite controllers, it could be useful clarify if in this study population, this kind of patients are present or not (according to my understanding not)

- I suggest to write in the main text the descriptive variables for all the patients, and to leave the differences among the two populations only in the table 1.

- I suggest to clarify at which timepoint the HIVDNA test was performed: at time of enrolment in Apache study? It was repeated?

- The authors stated that HLA-B27 and B57 are well documented and associated to HIV outcomes. I suppose that their prevalence is described in Figure 1 (it is not readable, it is too small) but also a comment in the text should be added. E.g. if some associations has been found also with this two HLAs.

- In the discussion, when HLA-B55 is mentioned, some previous data of other cohorts (references 17,18) seem to be in contrast with the findings of this study. Please give a comment on this.

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Reviewer #1: 1) The three reported signals are 21/78 vs. 1/18 (p=0.105), 1/78 vs. 3/16 (p=0.028), and 3/78 vs. 3/18 (p=0.087). If consider multiple testing adjustment, none is significant. Overall, there is no statistical power the declare any significant signal for the current study. More data or confirmation studies are needed.

The issue raised by the Reviewer is important and we agree on the fact that the study is not adequately powered to draw firm conclusions. However, not only fully-powered studies have been published in the scientific literature but also exploratory, proof-of-concept or pilot studies; despite these studies are characterized by a limited sample size, they have generally the merit to explore new scientific hypotheses.

We believe that the study design, the methods applied, the wording of our manuscript is in line with the small sample size; in fact, we proposed our work as an exploratory study, we conducted univariate analyses only, we stated that HLA-B39 and -B55 might be associated with HIV-1 DNA <100 copies/106PBMCs, we acknowledged among the limitations the small number of available patients and we concluded that the presence of HLA-B39 and -B55 may be associated with a lower level of total HIV-1 DNA, highlighting the need to deeply evaluate the role of HLA class I in affecting the size of reservoir.

According to the Reviewer’s comment, we also included in the discussion two additional considerations on these issues.

Reviewer #2: This aimed at exploring the correlation between low levels of HIV-1 DNA and specific HLA alleles. Although results should be confirmed in a larger sample size, the study is interesting and clearly reported and discussed. However there are some minor points to be addressed:

- Please, report in more details the methodology used to assess the quantification of HIV-DNA and of residual viremia. This part can be also included in the supplementary material.

According to the Reviewer’s comment, we added more details in the method section on residual viremia quantification and we also included a specific reference for HIV-DNA quantification where the assay used for HIV-DNA is clearly explained (De Rossi A et al. Quantitative HIV-1 proviral DNA detection: a multicentre analysis. New Microbiol. 2010; 33:293-302).

- Please, specify if the assay allows to quantify total or integrated HIV-DNA.

We specified in the method that we quantified total HIV-DNA.

- Please, better specify the sentence “HIV-1 RNA was <50 copies/mL for a median time of 11.7 (10.6-14.0) years and absence of plasma residual viremia for 6.9 (6.2-7.2) years”. Does it mean that HIV-1 RNA or residual viremia was persistently undetectable or absent throughout the median time reported, with no episodes of viral blips?

Thanks for the comment; we added in the method section “without any viral blips” to better clarify the inclusion criteria of the study.

- Please, modify “Nadir CD4+ were” in “Nadir CD4+ T cell count was”

Thanks for the suggestion, we changed “Nadir CD4+ were” in “Nadir CD4+ T cell count was” in the text.

- Which was the selection criteria for the 88 patients screened for HLA?

Among 96 participants screened for HIV DNA, we have available HLA data for 88 patients.

- In the footnotes of Table 1, please report the statistical tests used to assess statistical significance.

According to the Reviewer’s comment, Table 1 now includes the statistical test applied to compare the two groups of patients.

- In Figure 1, please highlight those HLA alleles significantly associated with different levels of HIV-1 DNA.

We modified Figure 1 according to the Reviewer’s comment.

- Is there a correlation between HLA and the outcome of analytical treatment interruption?

We explored a possible correlation between HLA and the outcome of analytical treatment interruption but we did not find any association.

Reviewer #3: I suggest to better clarify some aspects:

- More information about study population should be useful: year of enrolment, type of regimens and line of ARV, if they ever failed… Moreover, since the discussion includes consideration about LTNP and elite controllers, it could be useful clarify if in this study population, this kind of patients are present or not (according to my understanding not)

According to the Reviewer’s comment, we added the requested information into the manuscript. The exploratory study here reported is a parent study of the APACHE trial that excluded LTNP and elite controllers (enrolled patients had to stop ART); so, we clarified this issue in the methods of the manuscript.

- I suggest to write in the main text the descriptive variables for all the patients, and to leave the differences among the two populations only in the table 1.

As suggested, we removed the overall column from the table and included in the text some overall patients’ characteristics.

- I suggest to clarify at which timepoint the HIVDNA test was performed: at time of enrolment in Apache study? It was repeated?

As reported in the manuscript, HIV-DNA was performed to screen participants of the APACHE study; it was repeated only in the patients included in the APACHE study who underwent analytic treatment interruption (ATI). HIV-DNA trend during ATI remained below 100 copies/106PBMCs at all study visits except in two participants.

- The authors stated that HLA-B27 and B57 are well documented and associated to HIV outcomes. I suppose that their prevalence is described in Figure 1 (it is not readable, it is too small) but also a comment in the text should be added. E.g. if some associations has been found also with this two HLAs.

According to the Reviewer’s comment, we better clarified in the discussion section the role of HLA-B27 and HLA-B57 in our study (“In our analysis, these protective alleles were unexpectedly described with a low frequency among people with HIV-1 DNA <100 copies/106PBMCs and we did not find any correlation with the size of HIV-1 reservoir; further investigations on a larger sample size will be helpful to better interpret these findings.”)

Moreover, HLA-B27 was present in 5 participants (5.6%; 4 with HIV-DNA ≥100 copies/106PBMCs and 1 with HIV-DNA <100 copies/106PBMCs), while HLA-B57 in 4 (4.5%), all with HIV-DNA ≥100 copies/106PBMCs.

- In the discussion, when HLA-B55 is mentioned, some previous data of other cohorts (references 17,18) seem to be in contrast with the findings of this study. Please give a comment on this.

Thanks for the suggestion, we made a comment in the discussion section about these data (“multicenter studies conducting on a greater number of participants should be designed to clarify the role of HLA-B55 in HIV-1 infection”).

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1 Mar 2022

Association between low levels of HIV-1 DNA and HLA class I molecules in chronic HIV-1 infection

PONE-D-21-31418R1

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Additional Editor Comments (optional):

The paper is suitablle  for publication, as the concerns raised by the reviewer #1, namely that the study is underpowered and it doesn't add any value to clarify this controversial topic, did not undermine the conclusions drawn in the paper presented as an exploratory study.

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Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #3: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

Reviewer #3: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #3: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #3: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #1: Yes

Reviewer #3: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The relationship between HLA-B39 and rival replication is controversial in literature, as shown in the first paragraph of Discussion, and the current study doesn't add any value to clarify the controversy. If it were the first study to explore the relationship, it may have some values. Being aware of the controversy, one should design a study to solve the problem instead.

Reviewer #3: (No Response)

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Reviewer #1: No

Reviewer #3: No

7 Mar 2022

PONE-D-21-31418R1

Association between low levels of HIV-1 DNA and HLA class I molecules in chronic HIV-1 infection

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