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. 2020 Aug 6;15(8):e0237062. doi: 10.1371/journal.pone.0237062

Nutritional status positively impacts humoral immunity against its Mycobacterium tuberculosis, disease progression, and vaccine development

Mamiko Niki 1, Takashi Yoshiyama 2, Hideaki Nagai 3, Yuji Miyamoto 4, Makoto Niki 1, Ken-ichi Oinuma 1, Taishi Tsubouchi 1, Yukihiro Kaneko 1, Sohkichi Matsumoto 5, Yuka Sasaki 2, Yoshihiko Hoshino 4,*
Editor: Frederick Quinn6
PMCID: PMC7410285  PMID: 32760105

Abstract

Nutritional status contributes to the regulation of immune responses against pathogens, and malnutrition has been considered as a risk factor for tuberculosis (TB). Mycobacterium tuberculosis (Mtb), the causative agent of TB, can modulate host lipid metabolism and induce lipid accumulation in macrophages, where the bacilli adopt a dormant phenotype. In addition, serum lipid components play dual roles in the regulation of and protection from Mtb infection. We analyzed the relationship between nutritional status and the humoral immune response in TB patients. We found that serum HDL levels are positively correlated with the serum IgA specific for Mtb antigens. Analysis of the relationship between serum nutritional parameters and clinical parameters in TB patients showed that serum albumin and CRP levels were negatively correlated before treatment. We also observed reduced serum LDL levels in TB patients following treatment. These findings may provide insight into the role of serum lipids in host immune responses against Mtb infection. Furthermore, improving the nutritional status may enhance vaccination efficacy.

Introduction

Tuberculosis (TB) is one of the leading causes of death worldwide and remains a major global public health problem. According to the WHO Global Tuberculosis Report 2017, about 10 million people newly developed TB and 1.6 million died [1]. In addition, it was reported that 23% of the world population is latently infected with Mycobacterium tuberculosis (Mtb), the causative pathogen of TB, and is at high risk of Mtb reactivation.

As the nutritional status of the host plays a significant role in the maintenance of health status, the relationship between nutrition and infectious diseases has been widely studied [2–4]. In TB studies, the link of malnutrition and disease progression has long been recognized [5], and the TB incidence rate is higher in developing countries where malnutrition and bad hygiene deteriorate health. The relationship between nutrition and TB was examined using animal models in the mid-20th century [6]. Harries et al. reported that TB patients have a higher degree of under-nutrition compared to healthy hospital staffs [7]. Another study indicated that patients with under-nutrition at the time of TB diagnosis showed a 2-fold higher risk of death [8]. Although researches for many years have demonstrated the impact of nutrition on the progression and reactivation of TB, the association between host lipid profile and TB remains controversial. Some epidemiological studies showed that obesity is protective against TB [9, 10], while Mtb preferentially acquires host lipids in order to cause and maintain disease [11], and dietary cholesterol intake positively correlates with an increased risk of TB progression [12].

Mtb infection forms granuloma comprised of fibroblasts that surround Mtb-infected macrophages. Whole genome analysis revealed that progression of the infection within granuloma is accompanied by dysregulation of host lipid metabolism [13]. Mtb induces the differentiation of macrophages to lipid-loaded foamy cells, and Mtb acquires a dormancy-like phenotype [14, 15]. An in vitro study showed that Mycobacterium avium cells in lipid-laden macrophages cease division, and the withdrawal of lipids from the culture medium triggers the decline of lipid bodies in macrophages and replication of the bacilli [16]. These data indicate that lipid profile changes in hosts may affect the Mtb cellular metabolic status in macrophages and overall disease status.

As Mtb is an intracellular pathogen, the activation of cell-mediated immunity has long been believed as crucial for protection from TB [17]. However, accumulating experimental evidence suggests that humoral immunity can modulate the immune response to intracellular pathogens [18–20], and that humoral immunity acts as an important component of protective immune responses to Mtb [21]. Current research indicates that IgG antibodies obtained from TB patients promoted Mtb infection in alveolar epithelial cells, whereas IgA antibodies inhibited Mtb infection [22]. Other research showed that purified IgA obtained from human colostrum exhibited an inhibitory effect against Mtb infection in mice [23]. In addition, IgA-deficient mice showed increased susceptibility against Mycobacterium bovis infection [24]. In addition to offering some measure of protection, recent studies have highlighted the potential of serum IgA levels as a marker of Mtb infection. It has been reported that elevated serum IgA levels against the Mtb antigen lipoprotein Z are observed in individuals with latent TB infection [25]. IgA antibodies targeting other Mtb antigens such as ESAT-6, CFP10, Rv2031(Acr) [26] and lipoarabinomannan [27] are also detected in Mtb-infected patients. These findings suggest that serum IgA acts as an immunological marker of Mtb infection in the airway mucosa as well as a marker for the induction of mucosal immunity.

Based on these findings, many researchers have focused on the induction of humoral immunity by developing a new TB vaccine to replace BCG, whereas others are focusing on improving the efficacy of BCG. The phase 2b trial of M72/AS01E noted the induction of humoral immunity as well as cellular immunity and enhanced vaccine effectiveness [28]. On the other hand, several studies attempted to induce B cell responses against BCG by mucosal vaccination [29–31]. The importance of humoral immunity in TB vaccine development has been well reviewed elsewhere [21, 32, 33].

We previously evaluated the association between clinical status and serum antibody levels against several Mtb antigens in order to assess the potential of different antigens as novel vaccine candidates that elicit humoral immunity, and found that serum levels of Mtb antigen-specific IgA, not IgG, correlated with the positive clinical statuses of TB patients [34]. In this study, we analyzed whether the patients’ nutritional statuses affected the induction of specific isotypes of serum antibodies against Mtb. We also examined the relationship between serum nutritional parameters and clinical parameters in TB patients before and after treatment.

Subjects and methods

Participants

Patients of Fukujuji Hospital, and Tokyo Hospital, Tokyo Japan were consecutively enrolled after giving written informed consent. Patients were diagnosed with active-phase tuberculosis based on clinical symptoms, chest X-ray images, and bacterial cultures. When blood samples both before and after treatment were available, they were included in the analysis. A total of 22 patients in Fukujuji Hospital (age; 57.3 ± 14.8 yrs, males; 15, females; 7) were analyzed. All patients took Japanese standard medications for tuberculosis [35]. The following information was obtained from all patients at the time of enrollment: history of prior TB disease, work history in any healthcare setting or recent exposure to a patient with active TB, and other TB risk factors, such as having immunodeficiency disorders or taking immunosuppressive drugs. We used the same inclusion/exclusion criteria as in a previous study [34]. Information on previous medical history and clinical signs and symptoms were also collected as previously described. “The Japanese Society for Tuberculosis Classification” (1959) was applied to measure the severity of chest radiography at entry [36]. Briefly, tuberculosis lesions were classified by chest X-ray findings as type (cavity) and extent. “X-ray type (cavity)” was sub-divided from III to I (III: no cavity, II: morbid foci other than I, I: widespread cavities) and “X-ray extent” from 1 to 3 (1: minimal, 2: moderate, 3: severe). “Smear at entry” (entry = point of diagnosis before treatment) indicates the number of acid-fast bacilli inspected in the sputum smear taken at entry. The severity was subdivided as 0 (no acid fast bacilli (AFB) on smear), ± (1–2 AFB per 300 field), 1+ (1–9 AFB per 100 field), 2+ (more than 10 AFB per 100 field), and 3+ (more than 10 AFB per field). Blood sample collection was performed before treatment and after treatment. Serum concentrations of C-reactive protein (CRP), albumin, high-density lipoprotein (HDL), low-density lipoprotein (LDL) and total cholesterol were measured using a TBA-2000FR chemical analyzer (Toshiba Medical Systems Corporation, Tochigi, Japan) in the Division of Hematology in Niigata University Hospital. The research protocol was approved by the Institutional Review Boards of Osaka City University Graduate School of Medicine, Osaka, Japan, Tokyo Hospital, Tokyo, Japan, Fukujuji Hospital, Tokyo, Japan, and by the Research Ethics Committee of the National Institute of Infectious Disease, Tokyo, Japan.

Measurement of serum antibody levels

Concentrations of IgG and IgA antibodies specific for Mtb were determined by ELISA using recombinant proteins as previously described with slight modification [34]. Ninety-six well microplates (Sumilon Type H, LMS, Tokyo, Japan) were coated with each recombinant antigen in bicarbonate buffer, pH 9.6 and were blocked with phosphate buffered saline (PBS) containing 0.05% Tween 20 and 5% skim milk. Human serum samples diluted 1:200 in PBS containing 0.05% Tween 20 and 0.5% skim milk were added in duplicate (IgG) or triplicate (IgA) to the antigen-coated wells. HRP-conjugated anti-human IgG or IgA antibodies were added at a 1:2000 or 1:1000 dilution, respectively. For the visualization of the reactions, 100 μL of SureBlue reserve-TMB was added to each well. The reactions were stopped by acidification and the absorbance of each reaction solution was measured at 450 nm using a Multiskan Spectrophotometer (Thermo Fisher Scientific, Yokohama, Japan). The results of the IgG-ELISA were expressed as absorbance at 450 nm, whereas results of the IgA-ELISA were expressed as ELISA-Index, S / (B+3SD), where S is the average OD value of the duplicate test samples and B+3SD corresponds to the average OD value of the duplicate negative controls (B) plus three times the standard deviation (SD) [26].

Reagents and recombinant protein preparation

pET-21b, pET-22b, Luria-Bertani (LB) medium and carbenicillin were obtained from Sigma (St. Louis, MO, USA); isopropyl-1-thio-beta-D- galactopyranoside and Ni-NTA agarose were obtained from Qiagen (Gaithersburg, MD, USA); skim milk was from Morinaga (Tokyo, Japan); horseradish peroxidase-conjugated anti-human IgG or IgA antibodies was from Dako (Carpinteria, CA, USA); and SureBlue reserve TMB microwell peroxidase substrate was obtained from KPL (Gaithersburg, MD, USA). Expression and purification procedures for recombinant mycobacterial antigens (ESAT-6, CFP10, MDP1, Ag85A, Acr, HBHA and HrpA) were described previously [34].

Statistical analysis

The Mann-Whitney U-test was used to compare IgG and IgA levels between two independent groups, whereas one-way ANOVA was used for the comparison of three or more unmatched groups. Spearman’s rank correlation coefficient was used to determine the correlation of two independent values among ELISA values, nutritional status values and the severity of clinical status values. Wilcoxon signed-rank test was used for comparison of before-after treatment data in TB patients. All analyses were performed using online statistics calculators (http://www.socscistatistics.com/tests/Default.aspx, http://vassarstats.net/index.html). The threshold of significance was set at p < 0.05.

Results

Changes of antibody levels during treatment

We evaluated whether TB treatment affects the serum antibody levels for various Mtb antigens among TB patients. Previously, we observed a significant decrease in IgG levels against Acr and HrpA [37]. In this study, we could not find any changes in antibody levels during treatment (Figs 1 and 2).

Fig 1. Comparison of IgG responses to Mtb antigens in active TB (ATB) patients before and after treatment.

Fig 1

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The levels of serum IgG in ATB patients against 7 antigens (ESAT-6, CFP-10, MDP1, Ag85A, Acr, HBHA, and HrpA) before treatment (labeled as “Before”) and after treatment (“After”) were analyzed by ELISA. Data shown are the average of triplicate experiments. There were no statistical changes in the levels of serum IgG against TB antigens before and after treatment.

Fig 2. IgA responses to Mtb antigens.

Fig 2

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The levels of serum IgA in ATB patients against 7 antigens (ESAT-6, CFP-10, MDP1, Ag85A, Acr, HBHA, and HrpA) before treatment (labeled as “Before”) and after treatment (“After”) were analyzed by ELISA. The results of the IgA-ELISA were expressed as ELISA-Index, as described in “Subjects and Methods”. Also, there were no significant differences in the levels of serum IgA against TB antigens before and after treatment.

Clinical parameters and antibody levels

In order to clarify the relationship between serum antibody quantity and the disease status, serum antibody levels were compared to clinical parameters. Consistent with our previous observation [37], IgG levels against HrpA before treatment showed a positive correlation with “Smear at entry” (Fig 3).

Fig 3. The levels of serum IgG against HrpA before treatment in “Smear at entry”.

Fig 3

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The levels of serum IgG against HrpA before treatment were compared to the severity in “Smear at entry” sub-grouped between 1+, 2+ and 3+. HrpA-IgG levels before treatment were found to have a positive relationship with “smear at entry” scores. **: p < 0.01.

CRP and serum nutritional parameters

We analyzed the association between serum CRP and serum nutritional parameters (HDL, LDL, total cholesterol and albumin) at the onset of treatment. We found that the serum CRP levels were negatively correlated with serum albumin levels (Fig 4A). Although we could not find a direct correlation between CRP and lipid parameters, analysis of the relationship among nutritional parameters indicated that serum levels of HDL, LDL and total cholesterol were found to have positive correlations with serum albumin levels in TB patients before treatment (Fig 4B). Interestingly, analysis of serum samples obtained after treatment revealed that all nutritional parameters tested in this study showed negative correlations with CRP (Fig 4C), and serum albumin level were positively correlated with LDL and total cholesterol levels (Fig 4D).

Fig 4. Comparison of CRP and serum nutritional parameters.

Fig 4

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(A) Correlation between serum CRP level and albumin in active TB patients before treatment. (B) The relationship between serum lipids and serum albumin levels in TB patients at the onset of treatment. (C) The relationship between serum CRP levels and nutritional parameters in TB patients after chemotherapy. (D) The relationship between serum lipid parameters and serum albumin levels in TB patients after chemotherapy.

Disease severity and serum parameters

We observed whether disease severity diagnosed by chest X-ray and sputum smear indexes was associated with serum parameters including serum CRP. As we previously reported [34], significant associations were seen between serum CRP levels before treatment and “X-ray extent” as well as “Smear at entry” indexes (S1A Fig). In addition, serum albumin levels showed negative relationships with “X-ray extent” and “Smear at entry” indexes (S2A Fig). However, we could not find any association between disease severity and lipid parameters. We also evaluated whether the serum parameters after treatment correlated with clinical status at the onset of treatment. As expected, serum CRP levels showed a positive correlation with disease status. Additionally, we found a similar relationship between serum albumin levels and disease status (S1B and S2B Figs). On the other hand, there was no relationship between lipid parameters after treatment and disease status.

Correlation between antibody levels and nutritional status before and after treatment

In order to clarify whether nutritional status affected the host immune response, antibody levels were compared to nutritional parameters. Surprisingly, serum levels of HDL and total cholesterol showed positive relationships with serum IgA levels against Mtb antigens before treatment (Fig 5A and 5B). On the other hand, no correlations were observed between serum antibody levels and serum nutritional parameters after treatment.

Fig 5. Correlation between antibody levels and nutritional status before and after treatment.

Fig 5

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(A) Correlation between serum HDL concentrations and serum IgA levels specific to Mtb antigens before TB treatment. (B) Correlation between serum total cholesterol levels and serum IgA levels specific to Mtb antigens before TB treatment.

Change in serum nutritional status during treatment

Finally, we analyzed whether TB treatment affected the serum levels of nutritional parameters. Although we could not find any differences in levels of serum HDL, total cholesterol and albumin during treatment, a significant decrease was observed in serum LDL levels after treatment (Fig 6).

Fig 6. Changes in serum nutritional status in ATB patients during treatment.

Fig 6

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Analysis of serum nutritional levels showed a significant decrease in serum LDL levels in ATB patients after treatment. **: p < 0.01.

Discussion

In this study, we evaluated the relationship between immune responses against Mtb infection and host nutritional status. It has been reported that elevated CRP levels and decreased albumin levels are often seen in many inflammatory diseases, and are correlated with disease severity and mortality rate [38–41]. Artero et al. reported that hypoalbuminemia is a risk factor for mortality in patients with community-acquired bloodstream infections with severe sepsis [42]. In addition, recent studies indicated that serum albumin levels were decreased in HIV-infected TB patients [43] and were affected by antiretroviral and anti-TB therapy [44]. Consistent with these reports, we found a negative correlation between serum albumin level and CRP level in active TB patients before treatment. Although we could not find statistical significance between CRP and serum lipid levels, patients with low serum albumin levels also exhibited low serum lipid levels before treatment. Interestingly, analysis of serum samples obtained from TB patients after treatment revealed negative correlations between CRP and serum lipid parameters as well as serum albumin level. The role of serum lipids on inflammation during Mtb infection was not fully understand in this study because of the limitation of sample size and further study will be needed.

To clarify the role of serum nutritional status in the immune response against Mtb infection, we evaluated the correlation between serum nutritional status with Mtb-specific antibody profiles. We found a positive relationship between serum lipid parameters and the levels of Mtb antigen-specific IgA, suggesting that serum lipid components may enhance the mucosal immune response during Mtb infection. We previously reported that the presence of HDL cholesterol helps the internalization of Mtb into macrophages [45]. The spread of Mtb infection at the lung mucosa may trigger the rapid activation of the mucosal immune response, leading to elevated Mtb-specific IgA production. IgA in its secretory form is regarded as the main effector molecule of the mucosal immune system and serves as the first line of defense against pathogen invasion initiated at mucosal surfaces. One study conducted among healthcare workers with suspected latent TB infection (LTBI) patients showed that elevated serum IgA levels were seen in IFN-𝛶-positive subjects, suggesting a protective role of IgA in LTBI [46]. It was also reported that IgG and IgA against mycobacterial glycolipid antigens were found to be higher in QFT-positive LTBI healthcare workers [47]. A recent study by Zimmermann et al. revealed that inhibitory activity of antibodies against Mtb infection is dependent on their isotype. They found that purified serum IgG obtained from TB patients promoted the uptake of Mtb cells by A549 human lung epithelial cells, whereas serum IgA reduced the bacterial load. These results imply that elevated serum HDL cholesterol may be beneficial to Mtb-infected individuals for the proliferation of IgA-producing plasma cells at the early stage of infection.

Low effectiveness of BCG vaccination in elderly people has been one of the most serious problems in preventing the spread of pulmonary TB. In addition to the development of new vaccines to replace BCG, researchers have also been studying ways to enhance the efficacy of BCG. Recent studies have highlighted the importance of antibody-mediated immunity against Mtb infection [32, 48], as it is now understood that antibodies can confer protection against intracellular pathogens via Fc-receptor mediated phagocytosis [18, 49]. Therefore, improved vaccination strategies that modulate the humoral immune response are being actively studied. Since the association between nutrition and immune function has received considerable attention for many years [50, 51], nutritional status is very likely to affect vaccine efficacy [52]. It has also been reported that nutritional status affected IgA responses against various vaccinations, for example, 23 valent pneumococcal capsular polysaccharide vaccine, Pneumo‐vax and the Human Diploid‐Cell Rabies Vaccine [53–56]. Monitoring nutrition may be a practical and low-cost way to impact vaccination outcome.

Several studies indicated that low concentrations of lipids are often seen in patients with active TB, and that this correlates with disease severity [57, 58]. In addition, Akpovi et al. reported that restoration of serum HDL and total cholesterol levels was observed during treatment [59]. Contrary to the previous reports, we did not identify any changes in HDL and total cholesterol serum levels. The different results obtained in our study may be due to the nutritional status in Japan. According to the nutrition survey in 2010, dietary intake of cholesterol for Japanese adults >20 years old was in the range of 300 to 349 mg/day, which was higher than global mean dietary cholesterol intake (228 mg/day) [60]. On the other hand, we observed a significant decrease in serum LDL levels during treatment. Moreover, serum LDL levels were found to be lower in LTBI individuals than in active TB patients. It has been reported that Mtb infection induces lipid-droplet formation in macrophages [61]. The lipid-laden macrophages allow mycobacterial cells to persist and become defective in bactericidal activity [16, 61, 62]. LDL is known to be a main lipid acquisition source of foamy macrophage and the increased expression of CD36 and LOX1, macrophage surface receptors of oxidized LDL, was observed during mycobacterial infection [63, 64]. Although further studies are needed to solve the complicated relationship between serum lipids and TB progression, decreased LDL levels in patients after treatment may be a trigger of switching Mtb cells from an actively-growing state to a slow-growing dormant state in macrophages.

Conclusion

To our knowledge, this is the first human study to investigate the relationship between nutritional status and humoral immunity in a Japanese population. Serum albumin levels were negatively correlated with CRP at the onset of treatment. It was also found that serum albumin levels were positively associated with serum lipid levels. Evaluation of serum antibodies and nutritional parameters revealed that enhanced serum IgA specific to Mtb antigens was observed in TB patients with high serum HDL levels before TB treatment. On the contrary, serum LDL levels were decreased in both TB patients after treatment and LTBI individuals. The data in this study may provide an insight into the role of serum lipids in host immune responses against Mtb infection as well as in the acquisition of a dormancy-like phenotype by Mtb cells. The control and maintenance of optimal nutritional status may improve the outcome of vaccination against TB.

Supporting information

S1 Fig. Association between serum CRP levels and disease severity.

(A) Correlation between serum CRP before treatment and disease severity at onset. (B) Correlation between serum CRP after treatment and disease severity at onset. For statistical analyses, groups I and II of “X-ray cavity” and groups 1 and 2 of “Smear at entry are combined because of the small sample number of patients diagnosed as group I in “X-ray cavity” and 1 in “Smear at entry”. **: p < 0.01, *: p < 0.05.

(TIFF)

Click here for additional data file. (459.8KB, tiff)
S2 Fig. Association between serum albumin levels and disease severity.

(A) Correlation between serum albumin before treatment and disease severity at onset. (B) Correlation between serum albumin after treatment and disease severity at onset. **: p < 0.01, *: p < 0.05.

(TIFF)

Click here for additional data file. (458KB, tiff)

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

The authors are thankful to all healthcare workers and tuberculosis patients who participated in this study. This work was supported in part by AMED under Grant Number JP18FK0108075 (to Y. H.) and by a Grant-in- Aid for Scientific Research (C) from the Japan Society for the Promotion of Science for Mam. N. (16K09584) and Y. H. (18K08312). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Decision Letter 0

Frederick Quinn

12 Nov 2019

PONE-D-19-26473

Nutritional status positively impacts humoral immunity against its Mycobacterium tuberculosis, disease progression, and vaccine development.

PLOS ONE

Dear Dr. Hoshino,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

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We look forward to receiving your revised manuscript.

Kind regards,

Frederick Quinn

Academic Editor

PLOS ONE

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Reviewers' comments:

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Comments to the Author

1. 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: Yes

Reviewer #2: Partly

**********

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

Reviewer #1: I Don't Know

Reviewer #2: No

**********

3. 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: Yes

Reviewer #2: Yes

**********

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

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. 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: Thank you for submitting the manuscript. There are few questions/ comments that will help clarify few things

1. Please provide a reason not to include nutritional data from patients used in your earlier published studies. This would increase the number of patients and benefit the statistical analysis.

2. Please provide more demographic details about the 22 patients enrolled in this study

3. Please consider either adding more LTBI patients or removing those patients from the study. Only 6 data points is not enough to come to any conclusion.

4. IS there any correlation between serum albumin or HDL versus TNFa levels in active TB patients?

5. Please provide Fig 5B

Reviewer #2: Firstly and most importantly, the topic is too big for a small scale of study. As stated, 22 patients and 6 patients from 2 hospital, may not be sufficiently supportive for the correlation study. I also doubt about the statistical analysis for such small number of patients. I strongly recommend the authors to perform the study on a larger scale, on the number of both medical centers and patients.

Secondly, a lot of the results and conclusions are based on only observational and descriptive data. For example, it’s pretty easy to perform such study, by describing what data and thus correlation from such results. However, how to further confirm and verify, still remains unclear in this study. For example, could the authors provide evidence on the correlation as concluded, by changing the lipid level then obtaining the change in TB development?

Thirdly, one slight drawback is, since the authors had stated the nutritional status, however, as seen from the section of Materials and Methods, the authors only stated all patients will follow the standard treatment in Japan for TB. Then how about the diets, which will eventually change their nutritional status? Consequently, detailed description are required to support the results and conclusions.

**********

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Reviewer #2: No

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PLoS One. 2020 Aug 6;15(8):e0237062. doi: 10.1371/journal.pone.0237062.r002

Author response to Decision Letter 0

7 May 2020

30th April, 2020

Professor Fredrick Quinn,

Academic Editor of PLOS ONE

Re: PONE-D-19-26473

Dear Prof. Fredrick Quinn,

We appreciate the opportunity to revise our original manuscript entitled “Nutritional status positively impacts humoral immunity against its Mycobacterium tuberculosis, disease progression, and vaccine development.”. The original manuscript has been dramatically modified according to the reviewers’ comments and suggestions, and we believe the revised manuscript will be more informative for readers of your journal.

Our responses to the reviewers’ comments are addressed in detail below.

Again, we are grateful for the opportunity to submit a manuscript for publication in PLOS ONE.

Sincerely Yours,

Yoshihiko Hoshino, MD, PhD

Department of Mycobacteriology National Institute of Infectious Diseases, Higashi-Murayama, Tokyo, JAPAN

We wish to thank the reviewer for the insightful comments and suggestions on our paper. The comments have helped us improve the paper.

1. Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified (1) whether consent was informed and (2) what type you obtained (for instance, written or verbal, and if verbal, how it was documented and witnessed). If your study included minors, state whether you obtained consent from parents or guardians. If the need for consent was waived by the ethics committee, please include this information.

Informed consent was obtained as a written form. We added the information in line 101, page 6.

2. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

In accordance to the reviewer #1’s suggestion, we have removed the data obtained from LTBI patients and the phrase “data not shown” that refers to the data corresponding to the serum nutritional levels of LTBI patients.

Answers to the comments

Reviewer #1

We strongly appreciate the reviewer’s comments and suggestions on this paper.

Answers to the specific comments:

1. Please provide a reason not to include nutritional data from patients used in your earlier published studies. This would increase the number of patients and benefit the statistical analysis.

As the blood sample volumes obtained from the patients were low and limited, we could not afford to determine the serum biochemical parameters in addition to the analysis of serum antibody levels. In this study, we collected only the samples with enough volume for the measurement of serum nutritional status and the antibody levels at the same time.

2. Please provide more demographic details about the 22 patients enrolled in this study.

In accordance to the reviewer’s comment, we have added the demographic details about the patients to the line 104, page 6.

3. Please consider either adding more LTBI patients or removing those patients from the study. Only 6 data points is not enough to come to any conclusion.

According to the reviewer’s comment, we have removed LTBI patients from this study.

4. IS there any correlation between serum albumin or HDL versus TNFa levels in active TB patients?

We agree to make an analysis between HDL and inflammatory cytokines such as TNFa. Unfortunately, we have not determined the serum TNF-alpha levels in this study. As stated in 1, we only collected small volume of samples.

5. Please provide Fig 5B.

We are sorry not including Fig 5B now we have attached Fig 5B in the file.

Reviewer #2

Firstly and most importantly, the topic is too big for a small scale of study. As stated, 22 patients and 6 patients from 2 hospital, may not be sufficiently supportive for the correlation study. I also doubt about the statistical analysis for such small number of patients. I strongly recommend the authors to perform the study on a larger scale, on the number of both medical centers and patients.

We agree to the reviewer #2. If we could expand the numbers however, we are afraid we could not, which is a limitation of this study.

Secondly, a lot of the results and conclusions are based on only observational and descriptive data. For example, it’s pretty easy to perform such study, by describing what data and thus correlation from such results. However, how to further confirm and verify, still remains unclear in this study. For example, could the authors provide evidence on the correlation as concluded, by changing the lipid level then obtaining the change in TB development?

We really agree to the reviewer. However, this is just observational and descriptive study. Further study and analysis should be required, which we completely agree.

Thirdly, one slight drawback is, since the authors had stated the nutritional status, however, as seen from the section of Materials and Methods, the authors only stated all patients will follow the standard treatment in Japan for TB. Then how about the diets, which will eventually change their nutritional status? Consequently, detailed description are required to support the results and conclusions.

As far as diets, we did not evaluate these items. All patients were given regular meals. No special diets were considered.

Attachment

Submitted filename: PLoS+ONE+response.docx

Click here for additional data file. (18.8KB, docx)

Decision Letter 1

Frederick Quinn

4 Jun 2020

PONE-D-19-26473R1

Nutritional status positively impacts humoral immunity against its Mycobacterium tuberculosis, disease progression, and vaccine development.

PLOS ONE

Dear Dr.Hoshino,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

  • A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

  • An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Frederick Quinn

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

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: (No Response)

Reviewer #2: (No Response)

**********

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: Partly

Reviewer #2: Partly

**********

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

Reviewer #1: I Don't Know

Reviewer #2: No

**********

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: Yes

Reviewer #2: No

**********

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

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

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: Thank you for the revisions. Here are few minor changes

1. Line 52 "obesity is protective against" please provide a reference.

2. Line 104 demographics - Since gender cannot be in fractions please provide the exact number of males and females in the study.

3. Line 113 – 115 – X ray results – Is it possible to keep the scoring system consistent for X ray type and extent? Keep it either in ascending or descending order? Similarly change the figure S1? It is otherwise very confusing.

4. Line 118 and 119 – It is not clear what is +2 and +3? Is it >10 AFB per 100x field for +2? For +3, please explain more than 10 AFB per field. What is per field?

5. Line 153 and 173 please provide the details of the statistical analysis applied to " Before" and "After" samples since they are paired.

6. Figure 1 and 2 - please position the "Before" label correctly on the graph.

7. Figure 2 - What is the rationale behind showing the IgG ELISA data as absorbance 450 nm and IgA ELISA data as ELISA index? Please consider a uniform data presentation.

8. Line 177 and 178 - please remove the p values since they are not indicated in the actual graph (fig. 2).

9. For Figure 1, 2 and 3 legends - Instead of writing what was done, it would help to write the results / observations.

10. Line 177 and 188 - for statistics please write either p or P . Keep it uniform.

11. Please provide figure 4C and 4D .

12. Line 202 - change TB patents to TB patients.

13. Line 216 (Fig S1)- There is a difference between serum CRP levels before and after treatment. Just add a line stating that this is as expected. With decrease in disease severity with treatment, the serum CRP levels will decrease too.

14. Please provide Figure 5B

Reviewer #2: As seen from the response, the authors had barely made revision according to my comments. Consequently, I have to stick with the rejection decision. Thanks.

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2020 Aug 6;15(8):e0237062. doi: 10.1371/journal.pone.0237062.r004

Author response to Decision Letter 1

7 Jul 2020

We wish to thank the reviewer for the insightful comments and suggestions on our paper. The comments have helped us improve the paper.

Answers to the specific comments.

1. Line 52 "obesity is protective against" please provide a reference.

→In accordance to the reviewer’s comment, we have added the references to the text.

2. Line 104 demographics – Since gender cannot be in fractions please provide the exact number of males and females in the study.

→We have provided the exact number of males and females to the text.

3. Line 113 – 115 – X ray results – Is it possible to keep the scoring system consistent for X ray type and extent? Keep it either in ascending or descending order? Similarly change the figure S1? It is otherwise very confusing.

→We completely agree with a reviewer however, this scoring system was established in 1959 and was long time used as a standard scoring system in Japan. Again, we agree that the system is a little bit curious and confusing but we cannot modify the system by ourselves without any permission.

4. Line 118 and 119 – It is not clear what is +2 and +3? Is it >10 AFB per 100x field for +2? For +3, please explain more than 10 AFB per field. What is per field?

→This is according to “diagnostic standards and classification of tuberculosis and other mycobacterial diseases (American Review of Respiratory Disease 1981, 123(3), 343–358). The scale was determined by Ziehl-Neelson staining, and per field means x1,000 field by an optical microscope (in the literature described above, there was an explanation as “Examination at 800-1000X is assumed”).

In conversion of the conventional Gaffky scale, + accounts for G1 (conventional Gaffky scale), 1+; G2, 2+; G5, 3+; G9.

5. Line 153 and 173 please provide the details of the statistical analysis applied to "Before" and "After" samples since they are paired.

→We have added the description about statistical analysis in the text.

6. Figure 1 and 2 - please position the "Before" label correctly on the graph.

→As requested, we have corrected the position of the “Before” labels in the graphs.

7. Figure 2 - What is the rationale behindshowing the IgG ELISA data as absorbance 450 nm and IgA ELISA data as ELISA index? Please consider a uniform data presentation.

→We thank the reviewer’s comment on this point. Since we had to measure the antibody levels to multiple Mtb antigens using samples with limited volume, we evaluate IgG levels at the serum concentration as little as possible to save the samples. However, serum IgA levels are much lower than IgG when measured by the same method for the identification of serum IgG levels, we introduced the ELISA Index to evaluate the IgA levels in our previous study and the present manuscript according to the previous reports describing similar experiments. We have added the references to the text.

8. Line 177 and 178 - please remove the p values since they are not indicated in the actual graph (fig. 2).

→We have removed the p values from the legend.

9. For Figure 1, 2 and 3 legends - Instead of writing what was done, it would help to write the results / observations.

→As suggested, we have added the brief descriptions about the results to the legends.

10. Line 177 and 188 - for statistics please write either p or P. Keep it uniform.

→In accordance to the reviewer’s comment, we standardize the description of p-values in the text. Accordingly, we have also changed the “P” value to “p” in Fig. 3.

11. Please provide figure 4C and 4D.

→We appreciated this comment and corrected as the attachment.

12. Line 202 - change TB patents to TB patients.

→We have corrected the typo.

13. Line 216 (Fig S1)- There is a difference between serum CRP levels before and after treatment. Just add a line stating that this is as expected. With decrease in disease severity with treatment, the serum CRP levels will decrease too.

→In accordance to the reviewer’s suggestion, we have changed the text as follows: As expected, serum CRP levels showed a positive correlation with disease status. Additionally, we found a similar relationship between serum albumin levels and disease status (S1B and S2B Figs). On the other hand, there was no relationship between lipid parameters after treatment and disease status.

14. Please provide Figure 5B

→We appreciated this comment and corrected as the attachment.

Attachment

Submitted filename: Point by Point_200630_rev3.docx

Click here for additional data file. (17.4KB, docx)

Decision Letter 2

Frederick Quinn

21 Jul 2020

Nutritional status positively impacts humoral immunity against its Mycobacterium tuberculosis, disease progression, and vaccine development.

PONE-D-19-26473R2

Dear Dr. Hoshino,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Frederick Quinn

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

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 #2: 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 #2: Yes

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

Reviewer #2: Yes

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Reviewer #2: Yes

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PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

**********

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Reviewer #2: (No Response)

**********

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If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: Yes: Zhenbo Xu

Acceptance letter

Frederick Quinn

27 Jul 2020

PONE-D-19-26473R2

Nutritional status positively impacts humoral immunity against its Mycobacterium tuberculosis, disease progression, and vaccine development.

Dear Dr. Hoshino:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Frederick Quinn

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Fig. Association between serum CRP levels and disease severity.

    (A) Correlation between serum CRP before treatment and disease severity at onset. (B) Correlation between serum CRP after treatment and disease severity at onset. For statistical analyses, groups I and II of “X-ray cavity” and groups 1 and 2 of “Smear at entry are combined because of the small sample number of patients diagnosed as group I in “X-ray cavity” and 1 in “Smear at entry”. **: p < 0.01, *: p < 0.05.

    (TIFF)

    Click here for additional data file. (459.8KB, tiff)
    S2 Fig. Association between serum albumin levels and disease severity.

    (A) Correlation between serum albumin before treatment and disease severity at onset. (B) Correlation between serum albumin after treatment and disease severity at onset. **: p < 0.01, *: p < 0.05.

    (TIFF)

    Click here for additional data file. (458KB, tiff)
    Attachment

    Submitted filename: PLoS+ONE+response.docx

    Click here for additional data file. (18.8KB, docx)
    Attachment

    Submitted filename: Point by Point_200630_rev3.docx

    Click here for additional data file. (17.4KB, docx)

    Data Availability Statement

    All relevant data are within the manuscript and its Supporting Information files.

    Articles from PLoS ONE are provided here courtesy of PLOS

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