Multiple Infections, Recombination, and Hypermutation During a 12-Month Prospective Study of Five HIV-1 Infected Individuals

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In the manuscript, the authors have reported the analysis of viral sequences from five patients for the period of 12 months. They have carefully described the structure of viral genomes, provided some insight of the evolution of these genomes.   

Author Response

Comment 1: "In the manuscript, the authors have reported the analysis of viral sequences from five patients for the period of 12 months. They have carefully described the structure of viral genomes, provided some insight of the evolution of these genomes."

Response 1: Thank you.

Reviewer 2 Report

Comments and Suggestions for Authors

The present study describes a longitudinal analysis of HIV-1 genome instability in five infected individuals over a course of one year. The primary strength of the work lies in its dataset; however, this is outweighed by major concerns, including: (1) a lack of a novel conceptual advance beyond confirming known viral dynamics, (2) a descriptive Discussion that struggles to provide a meaningful interpretation of the results, and (3) a contradiction regarding the treatment status of the cohort.

Major concern: lack of novelty and inconclusive Discussion. The central finding—that HIV-1 exhibits intra-patient genetic diversity and dynamics—is a well-established principle in virology. The study confirms existing knowledge but does not articulate a novel mechanistic insight or a significant advancement to the field. The authors themselves refer to other studies that have already shown similar dynamics, which further undermines the novelty. This issue is compounded by a Discussion section that is primarily descriptive, listing observations without providing deeper interpretation. For example, when discussing RDP5 results, the variable recombination rates are noted but not explored in the context of host-specific factors. The authors should reframe the manuscript to highlight a specific contribution and strengthen the discussion by proposing testable hypotheses to explain their observations.

Critical Methodological Inconsistency: There is a direct contradiction regarding the treatment status of the patient cohort. The Methods state the participants were treatment-naive, yet the Results attribute defective DNA clones to antiretroviral therapy. This fundamental error must be clarified and corrected, as it undermines the validity of the authors' interpretations and conclusions.

Minor issue with group representativity. The clinical and demographic description of the patient cohort is insufficient. Reporting only the average age, CD4 count, and viral load without providing ranges and standard deviations limits the reader's ability to assess the heterogeneity of the study population. Given that factors like disease stage (as inferred from CD4 count) can influence viral evolution, providing this basic descriptive data is essential for interpreting the results.

Conclusion

While the dataset presented is substantial, the manuscript in its current form requires substantial revision. The priority is to resolve the contradiction in patient treatment status. Subsequently, the authors should reframe the study to define its novel contribution to the field, potentially by delving deeper into the causes of the observed mutation patterns, and provide a mechanistic, interpretive discussion. The substantial dataset presents a good foundation, but these fundamental issues must be addressed.

Author Response

We thank the Reviewer for their constructive and insightful feedback on our manuscript. We have carefully considered each point and have undertaken substantial revisions to address the concerns. Below is our point-by-point response and a summary of the significant changes made to the manuscript.

Comment 1: "Major concern: lack of novelty and inconclusive Discussion. The central finding—that HIV-1 exhibits intra-patient genetic diversity and dynamics—is a well-established principle in virology. The study confirms existing knowledge but does not articulate a novel mechanistic insight or a significant advancement to the field. The authors themselves refer to other studies that have already shown similar dynamics, which further undermines the novelty. This issue is compounded by a Discussion section that is primarily descriptive, listing observations without providing deeper interpretation. For example, when discussing RDP5 results, the variable recombination rates are noted but not explored in the context of host-specific factors. The authors should reframe the manuscript to highlight a specific contribution and strengthen the discussion by proposing testable hypotheses to explain their observations."

Response 1: We fully agree with the Reviewer that intra-patient HIV-1 diversity is a well-established principle. The core contribution of our study is not to report this phenomenon as novel, but to use a longitudinal, multi-mechanism approach to document and compare the relative and dynamic contributions of co-occurring evolutionary processes, specifically, multiple infections, intersubtype/intrapatient recombination, and APOBEC-driven hypermutation, within treatment-naive, recently infected individuals from a highly recombinant epidemic setting.

To reframe the manuscript and strengthen the discussion as suggested, we made the following key revisions:

• Introduction: We condensed redundant background paragraphs and reframed the introduction to conclude with a clear, specific objective. The focus is now explicitly on assessing the combined contribution of multiple evolutionary forces over time in a defined cohort, positioning the study as an integrative, longitudinal analysis rather than a report of a single, known phenomenon.
• Results: We added text contextualizing the evolutionary patterns within the individual clinical profiles (e.g., noting descriptive trends between viral load and recombination signals), setting the stage for a more interpretive discussion.
• Discussion (Major Revisions): We have completely restructured and strengthened this section:
  • Positioning the Study: We inserted a new paragraph at the beginning of the discussion's concluding section that explicitly acknowledges the study's limitations (small N, single fragment) and frames its value as a pilot, hypothesis-generating investigation: "Given the small sample size and the use of a single genomic fragment, the conclusions presented here should be viewed as illustrative rather than definitive. The study provides a pilot-level, individualized characterization of HIV-1 intra-host dynamics and proposes testable hypotheses for future research..."
  • Proposing Testable Hypotheses: In line with the Reviewer's request, we now propose specific, testable hypotheses. For example:
    • Regarding variable intrapatient recombination rates (RDP5 results): We suggest this variation may be linked to "host-specific conditions, such as the level of immune activation or the presence of multiple infecting strains," and state these are factors for future study.
    • We added a dedicated paragraph connecting evolutionary patterns to clinical parameters, stating: "To complement the evolutionary analyses, we also considered the clinical and immunological context of each patient... This exploratory observation merits further investigation in larger cohorts." 
      • Mechanistic Interpretation: We enhanced the discussion of results from tools like jpHMM and SimPlot++ by elaborating on what their algorithmic differences imply for detecting recombination in real-world, complex populations, moving beyond a simple comparison of outputs.
• Conclusion: The final paragraph was revised to clearly summarize the study's integrated, longitudinal perspective and its implications for understanding individualized viral evolutionary trajectories.

Comment 2: "Critical Methodological Inconsistency: There is a direct contradiction regarding the treatment status of the patient cohort. The Methods state the participants were treatment-naive, yet the Results attribute defective DNA clones to antiretroviral therapy. This fundamental error must be clarified and corrected, as it undermines the validity of the authors' interpretations and conclusions."

Response 2: We sincerely apologize for this critical error in the original text. The Reviewer is absolutely correct. The statement in the Results attributing defective clones to ART was a severe oversight from a previous draft. All participants in this study were strictly treatment-naive throughout the entire follow-up period, as correctly stated in the Methods.

Corrective Actions Implemented:

1. Results Section (3.2): We have removed the incorrect sentence "These defective clones occurred due to antiretroviral therapy (ART)..." and replaced it with a clear, corrected explanation: "Defective proviral genomes observed here should not be interpreted as treatment-associated products, as all individuals were ART-naive. Their origin is more plausibly explained by endogenous processes such as APOBEC-mediated hypermutation, polymerase errors, and stochastic replication dynamics."
2. Methods Section (2.1): We have reinforced this point for absolute clarity by adding: "All individuals were strictly treatment-naive throughout follow-up. We removed all previous phrasing that could be erroneously interpreted as suggesting antiretroviral-therapy-induced selective pressure."
3. Discussion Section: We added a clarifying statement in the hypermutation discussion to prevent any ambiguity: "Because all participants were treatment-naive, the presence of defective clones cannot be attributed to ART. Instead, they likely reflect intrinsic viral and host processes.” We adjusted the wording throughout the manuscript to avoid any ambiguity regarding this point.

This correction is fundamental, and we have thoroughly checked the entire manuscript to ensure no other statements contradict the treatment-naive status of the cohort.

Comment 3: "Minor issue with group representativity. The clinical and demographic description of the patient cohort is insufficient. Reporting only the average age, CD4 count, and viral load without providing ranges and standard deviations limits the reader's ability to assess the heterogeneity of the study population. Given that factors like disease stage (as inferred from CD4 count) can influence viral evolution, providing this basic descriptive data is essential for interpreting the results."

Response 3: We added a table with detailed clinical and demographic characteristics of the patient cohort, including means, standard deviations, and ranges for age, CD4 count, and viral load.

We believe these comprehensive revisions directly and effectively address the Reviewer's major and minor concerns. The manuscript now more accurately represents the study's data, clearly frames its scientific contribution, and provides a more robust and interpretive discussion. We are grateful for the feedback, which has significantly improved the quality of our work.

 

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript is devoted to a longitudinal analysis of the genetic diversity of HIV-1 in five newly infected patients from Sao Paulo during one year of follow-up. The authors took samples every three months, amplified and cloned a fragment of the pol gene (protease and part of reverse transcriptase), performed sequencing and subsequent phylogenetic analysis, evaluated the presence of recombinations using several software packages, and analyzed signs of APOBEC-mediated hypermutation. The main conclusion of the study is that all five patients have multiple infections, frequent recombination events, and individual hypermutation variants.

The strengths of the manuscript include the relevance of the topic and the longitudinal design of the study. The on-farm evolution of HIV, multiple infections, and recombination are directly related to the formation of drug resistance and variability of the virus in the population. The presence of several observation points throughout the year allows us to talk not only about the static picture, but also about the dynamics of the viral population, which is much less common in the literature. The methodological part is described in some detail: the conditions of PCR, the approach to cloning, the assembly algorithms, the list of software used and the main parameters of the analysis are presented. The use of several independent tools for detecting recombinations is positively assessed, which reduces the risk of artefact interpretations. The authors indicate sequence numbers in the database, refer to additional materials, and generally demonstrate a commitment to reproducibility of the results. The discussion fairly honestly formulated methodological limitations, including the small sample size and possible problems associated with bulk-PCR.

At the same time, the work has a number of limitations that significantly limit the generalizability of the conclusions. First of all, the sample of five patients is very small and, in fact, allows us to consider the study as a pilot or illustrative one. In addition, only a relatively short fragment of the genome (the pol region) is analyzed, which does not provide a complete picture of intraspecific evolution and recombination at the genome-wide level. There are ambiguities and contradictions in the text in places: for example, the length of the amplified fragment is indicated differently in different sections, which may raise questions from the reader about what data these or other conclusions are based on. Separately, attention is drawn to the wording that most of the detected clones are "defective" and that this is due to antiretroviral therapy, whereas in the methods section, patients are described as treatment-naive. In its current form, this logic looks opaque and requires either additional justification or wording adjustments.

The text of the manuscript would benefit from editorial revision. The introduction contains repetitions of the same ideas (mutagenesis, recombination, hypermutation of APOBEC) in very similar formulations, which makes the narration somewhat redundant. The final conclusions sometimes sound too general, given the scale of the study: the statements about the "dynamic nature of the genetic landscape" are generally true, but it is desirable to tie them more strongly to the specific observations of this particular small cohort, emphasizing the illustrative, hypothesis-forming nature of the results. The clinical part is also not fully used: data on exercise and CD4 are provided, but the relationship between specific evolutionary patterns (multiple infections, recombination, hypermutation) and clinical and immunological parameters is practically not discussed, even at a descriptive level.

Some of these shortcomings can be corrected quickly enough without reworking the study itself.

I recommend: (1) to bring uniformity to the description of the amplified fragment and clearly indicate which section and how long was used in all analyses; (2) to clarify or reformulate the theses concerning "defective clones" and the role of antiretroviral therapy in relation to this cohort; (3) to shorten and condense the introduction somewhat, removing verbatim repeats; (4) soften the generalizing conclusions by adding reservations about the small sample size and the pilot nature of the work; (5) carry out light linguistic and technical proofreading to eliminate typos and uniform spelling of terms.

In general, the manuscript gives the impression of a well-executed, but rather medium research in terms of scale and level of generalization. The methodological part and mastery of the tools look not bad; the main conclusions correspond to the presented data, provided they are formulated more carefully. Taking into account the comments, first of all concerning the clarity of the description, the consistency of the text and the "landing" of the conclusions to the actual volume of the material, the work can be considered for acceptance for publication.

Author Response

We sincerely thank Reviewer 3 for their thoughtful, balanced, and constructive assessment of our manuscript. We are grateful for the recognition of the study's strengths and for the clear, actionable recommendations provided. We have carefully addressed each point raised, and the manuscript has been substantially revised accordingly. Below is our point-by-point response detailing the actions taken.

Comment 1: "At the same time, the work has a number of limitations that significantly limit the generalizability of the conclusions. First of all, the sample of five patients is very small and, in fact, allows us to consider the study as a pilot or illustrative one. In addition, only a relatively short fragment of the genome (the pol region) is analyzed, which does not provide a complete picture of intraspecific evolution and recombination at the genome-wide level."

Response 1: We included these limitations in the Discussion of our manuscript.

Comment 2: "There are ambiguities and contradictions in the text in places: for example, the length of the amplified fragment is indicated differently in different sections, which may raise questions from the reader about what data these or other conclusions are based on." "I recommend: (1) to bring uniformity to the description of the amplified fragment and clearly indicate which section and how long was used in all analyses;"

Response 2: This is a crucial point for clarity and reproducibility. We have standardized the description throughout the manuscript.

• In the Methods (Section 2.2): We now explicitly and consistently define the fragment. The text states: "generating a 1.15 Kbp fragment spanning the protease gene and a portion of the reverse transcriptase (RT) gene."
• We have checked and ensured that all subsequent analyses (phylogenetic, recombination, hypermutation) refer back to this defined 1.15 Kbp pol fragment.

Comment 3: "Separately, attention is drawn to the wording that most of the detected clones are "defective" and that this is due to antiretroviral therapy, whereas in the methods section, patients are described as treatment-naive. In its current form, this logic looks opaque and requires either additional justification or wording adjustments." "I recommend: (2) to clarify or reformulate the theses concerning "defective clones" and the role of antiretroviral therapy in relation to this cohort;"

Response 3: We apologize for this significant error and lack of clarity. The Reviewer is correct to highlight this contradiction. All participants were strictly treatment-naive.

• Correction in Results (3.2): We have removed the incorrect sentence linking defective clones to ART and replaced it with a corrected explanation: "Defective proviral genomes observed here should not be interpreted as treatment-associated products, as all individuals were ART-naive. Their origin is more plausibly explained by endogenous processes such as APOBEC-mediated hypermutation, polymerase errors, and stochastic replication dynamics."
• Reinforcement in Methods (2.1): We added: "All individuals were strictly treatment-naive throughout follow-up. We removed all previous phrasing that could be erroneously interpreted as suggesting antiretroviral-therapy-induced selective pressure."
• Clarification in Discussion: We added a note: "Because all participants were treatment-naive, the presence of defective clones cannot be attributed to ART. Instead, they likely reflect intrinsic viral and host processes. We adjusted the wording throughout the manuscript to avoid any ambiguity regarding this point."

Comment 4: "The text of the manuscript would benefit from editorial revision. The introduction contains repetitions of the same ideas (mutagenesis, recombination, hypermutation of APOBEC) in very similar formulations, which makes the narration somewhat redundant." "I recommend: (3) to shorten and condense the introduction somewhat, removing verbatim repeats;"

Response 4: We agree completely. The introduction has been significantly streamlined.

• We identified and removed two entire redundant paragraphs that near-verbatim repeated the description of mutation, recombination, and hypermutation.
• We replaced them with a single, concise paragraph that maintains the essential background while eliminating repetition: "HIV-1 genetic diversification results from high mutation rates, recombination events, and host-mediated editing processes such as APOBEC. These well-described mechanisms provide the foundation for understanding intra-patient viral evolution. To improve clarity, we removed redundant sentences and now present only the essential background necessary to contextualize the objectives of this study."
• This revision has greatly improved the flow and conciseness of the introduction.

Comment 5: "The final conclusions sometimes sound too general, given the scale of the study: the statements about the "dynamic nature of the genetic landscape" are generally true, but it is desirable to tie them more strongly to the specific observations of this particular small cohort, emphasizing the illustrative, hypothesis-forming nature of the results." "I recommend: (4) soften the generalizing conclusions by adding reservations about the small sample size and the pilot nature of the work;"

Response 5: This is a key recommendation that we have implemented throughout the manuscript's concluding sections.

• In the Discussion: We have inserted a new, explicit paragraph that directly addresses this point: "Given the small sample size and the use of a single genomic fragment, the conclusions presented here should be viewed as illustrative rather than definitive. The study provides a pilot-level, individualized characterization of HIV-1 intra-host dynamics and proposes testable hypotheses for future research involving broader genomic regions and larger cohorts."
• Throughout the Discussion: We have reframed language to be more precise. For example, broad statements about "all patients" are now more carefully tied to "in our five-patient cohort." We emphasize the value of the study in providing "illustrative insights" and "individualized characterization."

Comment 6: "The clinical part is also not fully used: data on exercise and CD4 are provided, but the relationship between specific evolutionary patterns (multiple infections, recombination, hypermutation) and clinical and immunological parameters is practically not discussed, even at a descriptive level."

Response 6: We thank the Reviewer for this excellent suggestion to better integrate our data. We have added this descriptive layer of analysis.

• In Results (3.1): After presenting the average clinical data, we added: "Although statistical associations cannot be inferred from this small cohort, descriptive examination suggests that individuals with higher viral loads tended to display higher recombination signal density, whereas participants with lower CD4 counts showed slightly increased frequencies of APOBEC-compatible mutations. These observations should be interpreted cautiously but help contextualize the evolutionary patterns within each patient’s clinical profile."
• In the Discussion: We added a dedicated paragraph to explore this connection: "To complement the evolutionary analyses, we also considered the clinical and immunological context of each patient. Although the cohort size precludes statistical conclusions, the qualitative patterns suggest that clinical parameters such as CD4 levels and viral load might influence the magnitude of recombination and hypermutation events. This exploratory observation merits further investigation in larger cohorts."

Comment 7: "I recommend: (5) carry out light linguistic and technical proofreading to eliminate typos and uniform spelling of terms."

Response 7: The entire manuscript has undergone thorough proofreading. We have corrected minor typographical errors, ensured consistent formatting of gene/protein names (e.g., APOBEC3G, Vif), standardized the presentation of units (e.g., bp, µL, copies/mL), and verified the consistency of terminology (e.g., "treatment-naive" vs. "ART-naive").

We believe that these revisions have directly addressed all of Reviewer 2's concerns, significantly strengthened the manuscript's clarity, precision, and scholarly framing. We are confident that the revised version presents a more accurate, careful, and impactful contribution. We thank the Reviewer again for their invaluable feedback.

Reviewer 4 Report

Comments and Suggestions for Authors

This original research article investigates viral evolution (genetic diversity, hypermutations and intersubtypic recombinations) in 5 HIV-infected individuals over a 12 month period in 5 visits. Researchers have used cutting edge bioinformatics for the study. I have only a few minor comments:  

1. In the Introduction section, first paragraph is repeated.
2. Names of primers, e.g., POL 1, INBO1R, K1 and others; what do they represent?
3. Data presented in Figures 2-6, and in Tables appears raw; it should be summarized and presented in some more intelligent way.
   1. 1.  

Author Response

We thank Reviewer 4 for their positive assessment of our work and their constructive minor comments. We are pleased that the use of cutting-edge bioinformatics was noted. We have addressed each point below.

Comment 1: "In the Introduction section, first paragraph is repeated."

Response 1: The Reviewer is absolutely correct. A paragraph in the original introduction was inadvertently duplicated. This has been corrected in the revised manuscript.

We have removed the redundant paragraph.

To ensure a smooth transition and maintain the necessary background, we replaced the two identical paragraphs with a single, concise statement: "HIV-1 genetic diversification results from high mutation rates, recombination events, and host-mediated editing processes such as APOBEC. These well-described mechanisms provide the foundation for understanding intra-patient viral evolution. To improve clarity, we removed redundant sentences and now present only the essential background necessary to contextualize the objectives of this study."

This revision eliminates the repetition while preserving the logical flow of the introduction.

Comment 2: "Names of primers, e.g., POL 1, INBO1R, K1 and others; what do they represent?"

Response 2: This is a valid point for improving methodological clarity. We have added an explanation in the Methods section.

In Section 2.2 (DNA Extraction, Quantification, and Amplification), immediately after listing the primer sequences for the first round of PCR, we have inserted the following clarification: "Primer names such as POL1, INBO1R, and K1 correspond to internal laboratory designations used to differentiate forward and reverse primers targeting the protease and partial reverse transcriptase regions of the HIV-1 pol gene."

This addition provides the necessary context for the primer naming convention without disrupting the technical description of the protocols.

Comment 3: "Data presented in Figures 2-6, and in Tables appear raw; it should be summarized and presented in some more intelligent way."

Response 3: We included the legend in Figures 2-6, and the colors are now clearer and more visible.  Furthermore, Panel B was placed below Panel A. In Table 1, we replaced the column 'S-E' with 'Start-End'. In Table 2, the asterisk is explained in the legend instead of at the end of the table. In Table 3, we combined columns S and E into a single Start–End column. Moreover, instead of listing entries such as “1114_V3_1, 1114_V3_2, …, 1114_V3_9,” we now use the condensed format “1114_V3_1–9.” In Table 4, the P-values are reported as percentages, and the legend states that all values are below 5%.