All of the 5 pulmonary lesions achieved partial response 6 months later, further shrank 1 year later, and were successfully controlled for 7 years. This case indicated that 125I seed implantation could be an alternative local therapy for metastatic pheochromocytoma in the lung.Pheochromocytoma with lung metastases is rare in clinics, and the prognosis of metastatic pheochromocytoma is generally poor. In this case, a 57-year-old woman who presented with hypertension and palpitations was diagnosed with left adrenal pheochromocytoma with lung metastasis in 2010. The patient received left adrenalectomy for pheochromocytoma 10 years ago, but pulmonary lesions had significant progression 7 years ago. The patient was treated with iodine-125 ( Pheochromocytoma is a rare endocrine neoplasm that arises from chromaffin cells of the adrenal medulla. Approximately 10% of pheochromocytomas are metastatic pheochromocytomas, and only metastases are the proof for malignant pheochromocytomas , 3. For 125I) seed brachytherapy is a modality of continuous low-dose-rate irradiation, in which a dose of <1 Gy per hour is continuously delivered by radioactive sources. 125I seed implantation is the most common approach of 125I seed brachytherapy and it has been performed in various types of locally recurrent or metastatic carcinomas in recent years  scans displayed a heterogeneous solid tumor, 6.9 × 5.5 cm in size, in the left adrenal medulla, as well as two nodules at less than 1 cm in the right lung . Three weeks before the surgery, the alpha-adrenergic receptor blocker, phenoxybenzamine (10 mg bid), was used for normalizing blood pressure and heart rate. The patient underwent left adrenalectomy to remove the tumor. The histopathological examination showed that the tumor was pheochromocytoma, and the Pheochromocytoma of the Adrenal Gland Scaled Score  to control blood pressure 2 weeks before the procedure and 2 months after the procedure. Percutaneous  mg bid tPheochromocytoma is a rare tumor in clinics, the incidence of pheochromocytoma is about 2 to 5 cases per million person-years . Nearly Surgical resection is the preferred treatment for patients with resectable metastatic pheochromocytomas, and a minimally invasive approach is recommended . However125I seed implantation was first used for the treatment of prostate cancer in the 1970s, and it has been the first-line treatment for localized prostate cancer  or 177Lutetium-DOTA-Tyr3-Octreotate (177Lu-DOTATATE), was recommended for the treatment of unresectable metastatic pheochromocytoma if tumors are positive on MIBG scan or somatostatin receptor imaging. As reported in a phase 2 multicenter trial  and gastrointestinal adverse events, such as nausea (78%) and vomiting (53%), were common. A study of 30 patients with metastatic or unresectable PPGL treated with 177Lu-DOTATATE displayed similar results  er trial  investig results . Partial results  showed, 125I seed implantation is a modality of internal radiation therapy, which releases continuous low-dose-rate x-ray and γ-ray. Although the type of radioactive rays and the form of irradiation are different from the radiopharmaceutical agents and EBRT, the pulmonary metastatic pheochromocytoma in our case showed good sensitivity to 125I seed radiation. Compared with targeted radiopharmaceutical agents and EBRT, 125I seed implantation provides the advantage of delivering higher doses of radiation to the tumor with relative sparing of surrounding normal tissues. The cumulative dose achieved 120-140 Gy in each pulmonary lesion in this case, while the median cumulative dose was only 24 Gy in EBRT  for the publication of any potentially identifiable images or data included in this article.WH conceived the idea of this case report and was responsible for the treatment. HS, WQ, and YY participated in the treatment. CW, BS, and HW participated in the follow-up and data collection. HS and CW wrote the first draft of the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Age is the major risk factor for mortality after SARS-CoV-2 infection and older people have received priority consideration for COVID-19 vaccination. However, vaccine responses are often suboptimal in this age group and few people over the age of 80 years were included in vaccine registration trials. We determined the serological and cellular response to spike protein in 100 people aged 80–96 years at 2 weeks after the second vaccination with the Pfizer BNT162b2 mRNA vaccine. Antibody responses were seen in every donor with high titers in 98%. Spike-specific cellular immune responses were detectable in only 63% and correlated with humoral response. Previous SARS-CoV-2 infection substantially increased antibody responses after one vaccine and antibody and cellular responses remained 28-fold and 3-fold higher, respectively, after dual vaccination. Post-vaccine sera mediated strong neutralization of live Victoria infection and although neutralization titers were reduced 14-fold against the P.1 variant first discovered in Brazil they remained largely effective. These data demonstrate that the mRNA vaccine platform delivers strong humoral immunity in people up to 96 years of age and retains broad efficacy against the P.1 variant of concern. The current COVID-19 pandemic has led to over 2.6 million deaths but approval and widespread administration of several COVID-19 vaccine platforms have led to hope that the current pandemic may be brought under control. However, in order for this to be achieved, it will be essential that vaccine-induced immune responses are elicited effectively in people of older age . The funA range of potential mechanisms may underlie the development of immune senescence, including a reduction in the number of naïve T cells due to thymic involution and accumulation of memory cells, as well as an increased serum concentration of inflammatory molecules in a phenomenon termed inflammaging . ApproacThe nucleoside-modified RNA vaccine BNT162b2 from Pfizer BioNTech incorporating spike (S) is strongly immunogenic but participants over the age of 75 years comprised only 4% of efficacy data . FurtherWe undertook an analysis of serological and cellular immune responses to spike protein in 100 independently living people aged between 80 and 96 years who received BNT162b2 vaccine with a 3-week interval between the first and second doses. We demonstrate strong humoral responses with evidence of broad neutralization of live Victoria virus and P.1 variants. Cellular immune responses were less marked and remained undetectable in 37% of donors.Serum samples were obtained from donors at 14–21 days following the second BNT162b2 vaccine (n=98). These were assessed initially for quantitative measurement of spike (S) responses using the Roche platform. S-specific responses were seen in all donors with 98% above 1 in 50. Two donors had low but positive titers of 1 and 2.5 and antibody responses were confirmed on the MSD platform .Nucleocapsid (N)-specific antibody serostatus was used to determine if donors had evidence of prior natural infection with SARS-CoV-2. This was seen in 10% of the cohort (10/98), with only 4 of these having reported previous symptoms indicating a high asymptomatic infection rate in this elderly cohort. Donors with prior SARS-CoV-2 infection had a median S-specific antibody titer of 32,250 which was 28-fold higher than the value of 1138 in those without prior infection (p<0.0001). No correlation was seen between the nucleocapsid titer and spike-specific titer amongst those with evidence of previous infection .Spike-specific ELISA (TBS) was also performed on eluates from dried blood spot (DBS) samples in order to assess the utility of the DBS platform in this age group . Very stTaken together, these results indicate strong spike-specific antibody responses develop in nearly all people aged above 80 years following the BNT162b2 vaccine regimen.Interferon gamma (IFN-γ) ELISpot analysis was then used to determine spike-specific T cell response following vaccination (n=98). Cellular responses against two peptide pools from the S1 and S2 spike domains, as well as membrane and nucleocapsid, were determined following overnight stimulation. Spike-specific T cell responses were detectable above threshold values in 63% of participants (62/98) and responses against the S1 and S2 domains were equivalent  . The medCellular responses were also assessed in relation to prior natural infection status. As expected, cellular responses against N were significantly greater in those with prior infection . Interestingly, this group also developed threefold stronger T cell responses against spike peptides after vaccination (228 spots/million PBMC) compared to uninfected people  . SimilarAlthough all of our donors were over 80 years of age, we were interested to assess if age remained a determinant of vaccine response within older people. Although our cohort contained people up to 96 years of age no correlation between age and humoral or cellular responses were observed against the spike protein . As suchAging can be associated with normal antibody levels but reduced functional activity and as such we next undertook live virus neutralization assays. Serial dilutions were performed to determine the reciprocal of the dilution that mediated 50% neutralization (ND50) with the Victoria and P.1 variant of concern n=20; . Assays Older people comprise a large proportion of the population in many countries and it is essential that COVID-19 vaccines provide protection in this vulnerable group. The potential importance of immune senescence in relation to covid vaccination remains uncertain although a negative influence of age has been seen in vaccinees aged up to 77 years .We detected spike-specific antibody responses in all older people which typically exceeded those seen after natural infection . It is lIn contrast, the induction of spike-specific cellular responses was less complete. T cell responses were present in 63% of people but were of relatively low magnitude. The S1 and S2 domains were equivalently immunodominant. However, it is noteworthy that undetectable or very low cellular responses were seen in 37% of people. The relative importance of cellular immunity in mediating clinical protection or sustaining humoral immunity is currently uncertain but these data indicate that this should be monitored prospectively. Indeed, a strong correlation was seen between the magnitude of the cellular and humoral immunity as observed in natural infection . It willPrimary SARS-CoV-2 infection presents a high clinical risk in people of this age but we observed that 10% of the cohort had evidence of prior infection. This was associated with substantially stronger humoral and cellular immunity after vaccination. Indeed, no participants with a history of previous natural infection had suboptimal cellular immunity compared to 44% in uninfected donors. Interestingly, we also observed strong responses after only the first vaccine dose in this group, with no significant increment after the second dose when administered with a 3-week interval. As such these findings are comparable with those seen in healthcare workers which have led to suggestions that a single vaccine delivery may be sufficient for those with prior natural infection .The optimal in vitro correlation of natural protection is assessment of live virus neutralization and strong neutralization of the Victoria strain correlated with global spike-specific response. The P.1 VOC contains a range of mutations within the spike domain including E484K which can mediate escape from recognition by some antibodies . A pronoOne of the limitations to this study includes the lack of pre-vaccination sample which is a reflection of the speed at which the vaccination program was rolled out in people over 80 years old and the challenge of operating within vaccine centers during national ‘lockdown.’ Future work should assess the longevity of the observed responses and neutralization of new variants that have emerged since the vaccination program started. This is now of great interest and may help to guide the need for further booster doses. Our work has focused solely on donors aged 80 years and older and, as such, it will also be important to see how immunity compares in younger cohorts who receive the BNT162b2 vaccine on a 3-week interval.In conclusion, our findings demonstrate that the BNT162b2 vaccine generates robust humoral responses in older people which is likely to underpin the clinical efficacy of this regimen. High levels of spike-specific antibody should ensure control of the Brazilian variant of concern in most people despite a 14-fold drop in neutralizing activity. Further work will be of interest to define immune correlates that may be used to guide approaches to maintain immune responses in the longer term.100 participants, aged 80 years or older, were recruited who were in living in community dwellings, able to attend a vaccination center, and did not require assistance with daily living or self-care. Written informed consent was obtained and the study was conducted according to the Declaration of Helsinki and good clinical practice. Participants were asked if they thought they had been infected with SARS-CoV-2 since the pandemic started.The median age of participants was 84 years (IQR 80–87 or range 80–96) and 58% were female. All participants received the Pfizer BNT162b2 COVID-19 vaccine with a 3-week interval between the first and second doses. A phlebotomy sample was taken at 2 weeks following the second vaccine. A finger prick DBS sample was also taken at 3 weeks following the first vaccination in 88 donors and at 2 weeks following the second vaccination .Antibodies specific to SARS-CoV-2 were detected using electrochemiluminescence assays on the automated Roche Cobas e801 analyzers based at Public Health England (PHE) Porton. Calibration and quality control were performed as recommended by the manufacturer. Anti-nucleocapsid protein (NP) antibodies were detected using the qualitative Roche Elecsys Anti-SARS-CoV-2 ECLIA , whilst total IgG, A, and M anti-spike (S) antibodies directed at the receptor-binding domain were detected using the quantitative Roche Elecsys Anti-SARS-CoV-2 S ECLIA , as previously described .Anti-nucleocapsid results are expressed as cutoff index (COI) value, with a COI value of ≥1.0 considered positive for anti-nucleocapsid antibodies. Anti-spike results are expressed as units per ml (U/ml), with samples with a result of ≥0.8 U/ml considered positive for anti-spike antibodies within the fully quantitative range of the assay: 0.4–2500 U/ml. Samples >2500 U/ml were diluted further  to within the quantitative range.Quantitative IgG antibody titers were measured against spike (S) protein, nucleocapsid protein (N), and other antigens using the MSD V-PLEX COVID-19 Respiratory Panel 2 )  from Meso Scale Diagnostics, Rockville, MD. Antigens were spotted at 200–400 μg/ml. Multiplex MSD assays were performed as per the instructions of the manufacturer. To measure IgG antibodies, 96-well plates were blocked with MSD Blocker A for 30 min. Following washing, with washing buffer, the samples were diluted 1:500 in diluent buffer. Reference standards and positive controls and diluted samples were added to the wells. After 2 hr of incubation and plates were washed 3× with wash buffer and detection antibody  diluted in diluent 100 was added. After 1 hr of incubation at room temperature (RT), the plates were washed 3× with wash buffer. MSD GOLD Read Buffer B was added and plates were read immediately using a MESO TM QuickPlex SQ 120. Text files were then generated from the Methodical Mind software then transferred to the MSD Discovery Workbench (v4.0) software. Data were then converted to AU/ml and exported as.csv files. The values from exported data were then adjusted for any sample dilution.×g for 10 min). The DBS eluate was then applied to a pre-coated 96-well ELISA plate  containing stabilized trimeric SARS-CoV-2 spike glycoprotein and detecting IgG, IgA, and IgM antibody isotypes. The performance characteristics for this assay were assessed in 162 non-hospitalized mild to moderate disease PCR-positive individuals and 707 presumed COVID-19 negative samples from pre-2019. Sensitivity was 96.3% (92.1–98.6) and specificity 99.3% (98.4–99.8). The ELISA output result was reported as a ratio relative to the cutoff calibrator and multiplied by the previously determined cutoff co-efficient to maintain batch-to-batch consistency, defined as 1.31 . Capillary blood samples were collected on DBS cards (Ahlstrom Munksjo) from participants remotely and stored at RT. Samples were eluted in 250 μl of 0.05% phosphate-buffered saline-Tween 20  per blood spot and incubated overnight (12–16 hr) before centrifugation  serum samples. SARS-CoV-2 was diluted to a concentration of 1995 pfu/ml (150 ffu/50 μl) and mixed 50:50 in 1% FCS/MEM with doubling serum dilutions from 1:20 to 1:640 in a 96-well V-bottomed plate (samples were further diluted where appropriate to get ND50s into the working range of the assay). The plate was incubated at 37°C in a humidified box for 1 hr to allow the antibody in the serum samples to bind the virus. Virus susceptible monolayers (Vero/E6 Cells) in 96-well plates were exposed to this serum/virus mixture. Plates were incubated in a sealed humified box for 1 hr before removal of the virus inoculum and replacement with semi-solid overlay (1% w/v CMC in complete media). The box was resealed and incubated for 20–24 hr prior to fixing for formaldehyde. Virus-specific foci were detected using a SARS-CoV-2 antibody specific for the SARS-CoV-2 RBD Spike protein and anti-rabbit HRP conjugate, infected foci were visualized using TrueBlueTM substrate. Stained foci were counted using ImmunoSpot S6 Ultra-V Analyzer (CTL) and resulting counts analyzed in SoftMax Pro v7.0 software. Values are stated as ND50, the reciprocal of the sample dilution causing 50% of mock-neutralized virus control.Quantification of IgG, IgA, and IgM was evaluated using the COBAS 6000 (Roche) at the University of Birmingham Clinical Immunology Service. About 29% of donors were found to have mild IgM deficiency (29/99), with 4% deficiency in IgA (4/99), and 1% in IgG (1/99).Xtend’ (Oxford Immunotec) and Ficoll. After quantification and dilution of recovered cells, 250,000 PBMCs were plated into each well of a ‘T-SPOT Discovery SARS-CoV-2’ (Oxford Immunotec) Kit. The kit is designed to measure responses to four different but overlapping peptides pools to cover protein sequences of four different SARS-CoV-2 antigens, without HLA restriction, and includes negative and positive controls. Peptide sequences that showed high homology to endemic coronaviruses were removed from the sequences, but sequences that may have homology to SARS-CoV-1 were retained. Cells were incubated and interferon-γ secreting T cells were counted. A cutoff of 6+ spots per million on the S1 pool was defined as a positive result in line with the Oxford Immunotec diagnostic COVID Kit.PBMCs were isolated from a whole blood sample using ‘T-Cell Data were tested for normality using Kolmogorov-Smirnov analysis. For comparative analysis of antibody titers following the first and second vaccines, a paired t-test was performed. For comparison of S1 and S2 ELISpot responses, Wilcoxon test was performed and for unpaired analysis NPE versus PE and comparison of variant neutralisation, Mann-Whitney was performed. Spearman rank correlation was used for comparing assay platforms for titers and correlating T cell responses to IgG titer. Source data file available for all figures. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:The paper tackles an important topic, the immune response to mRNA PfizerCOVID19 vaccination in the oldest group of the population, the 80 to 96 years old. The authors show high SARS-CoV2 antibody responses and robust cellular responses in this age group at 2 weeks following the second vaccination, which did not decrease from 80 to 96 years of age. Moreover, the authors demonstrate the use of dry blood spots assays for these analysis. In addition, the antibodies also show neutralization against the P1 variant, even though decreased as compared to the original strain.Decision letter after peer review:eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Jos van der Meer as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Debbie van Baarle (Reviewer #1).Thank you for submitting your article "BNT162b2 vaccination in people over 80 years of age induces strong humoral immune responses with cross neutralisation of P.1 Brazilian variant" for consideration by The reviewers have discussed their reviews with one another, and the Senior Editor has drafted this to help you prepare a revised submission.Essential revisions:1. The absence of a pre-vaccination sample makes the visualization of the vaccine response complicated. Moreover, visualization of a robust vaccine response and low pre-vaccination titers would strengthen the data. Why did the authors not include a pre-vaccination sample? This is also of importance with respect to point 3, the use of many different antibody tests2. The absence of comparison with a younger age group limits the use of the data presented in the paper. Even though robust antibody and cellular responses are shown in this age group, we don't know how that related to younger age groups and how 'impaired' immune responses are in this old age group. Do the authors have the possibility to compare their data to a younger age group? The data on cross-neutralization against the Brazilian variant is highly interesting. How does this difference in neutralization between the strains compares to younger age groups?3. The authors use many different antibody tests to analyze their data. How do these compare? What is the rational of using all these different tests? Are these tests standardized and validated to golden standards? Why do the authors use a different test for the measurement of the serum and DBS antibodies? This makes the comparison of serum to DBS complicated and less fair. The use of so many different tests is confusing, as are their read outs; do they all measure IgG, or what are differences? Please provide a rationale for this per figure and explain differences more clearly. This aspect really affects clarity of the paper and needs additional attention.4. The responses in the cellular tests are rather low with only 63% being positive. Which controls were included by the authors to investigate the robustness of the assay? These data are also rather limited by the lack of a pre-vaccination measurement. Do the authors have the possibility to compare to a pre-vac sample? As is done for the antibodies in Figure 1D. or to compare the results to a younger age group, to increase the reliability of the data shown.5. We feel that the correlation in Figure 1B is statistically wrong, due to the inclusion of so many negatives for nucleocapsid. We would suggest to analyze this differently, by just comparing the spike response in 2 groups: the N positive and N negative ones. A correlation can be made only in the N positive ones.6. Figure 1D: donors with previous exposure and no-previous exposure could be separated into two graphs to more clearly show that antibodies in those with prior exposure do not increase much after the second dose.7. Line 267 – the authors mention that they examined cellular responses against spike, membrane and nucleocapsid but only the responses against spike are shown in Figure 2. Lines 274-275 – the cellular responses against N are only mentioned in text. These data should be shown as figures.8. Why is the timepoint 2 weeks following vaccination chosen, and not 28 days post-vaccination as done in many clinical trials? How do the authors suggest that the responses do compare?9. How were the 20 donors down-selected for the neutralization assays in Figure 4? Was this randomized?10. Figure 4: it would be interesting to determine whether neutralization against a more neutralization-resistant variant (B.1.351 or B.1.617.2) is maintained in the elderly.11. The correlation between age and the humoral and cellular response is difficult to interpret. Given the restricted age range, it can be expected that there will be limited differences, unless there are major differences in health between these individuals. The title of this paragraph should be tuned down and the restriction of this analysis should be mentioned in the Discussion section.12. The Discussion section is somewhat limited. The authors should at least discuss the limitations of the study and indicate what additional information would be needed to draw firm conclusions.13. Please rephrase the sentences 74 to 80 in the introduction, which lacks proper understanding of immunological ageing.14. What do the authors mean by independently-living people as mentioned in sentence 87 in the introduction? Is that the proper terminology? Is community dwelling more appropriate?Reviewer #1 (Recommendations for the authors):15. Throughout the paper (including the title), the authors should avoid referring to the variants of concern with their country of origin. Either use the pango lineage names (P.1) or WHO's new Greek alphabet naming system for VOCs, in which P.1 is the γ variant.The authors tackle an important topic. It is important to analyze the immunogenicity of the mRNA vaccines in the oldest age group, the most vulnerable of the population. The notion of a robust immune response following vaccination is of importance for protection of this vulnerable age groups. Nevertheless, the authors did not compare the vaccine responsiveness to a younger age groups, to analyze the effect of age in these oldest olds. Moreover, the paper lacks a pre-vaccination sample. The authors show that the antibodies induced by the vaccination are also neutralizing against the Brazilian variant, a variant with a high rate of mutations in the spike protein, which is of importance to assess the potential threat of mutants on vaccine induced immunity. Nevertheless, the neutralization against the Brazilian variant is decreased as compared to the wild-type variant.It would be important to investigate the long-term immune response to the vaccination in this age group, to investigate how long the oldest can be protected by the vaccines. The authors here only show the immune response at the very short-term, 2 weeks after 2nd vaccination, which does not provide answers for the long-term.The authors provide an indication for the use of Dry blood spots for these kind of analysis and show a moderate correlation with the results found in blood. This analysis is however complicated by the use of different antibody assays. The authors found a moderate correlation between the cellular and antibody responses, but due to the high degree of variation in antibody assays used, this data is difficult the interpret.Reviewer #2 (Recommendations for the authors):Finally, the authors show a different vaccine response in the individuals that have been infected before vaccination. These participants were identified by the measurement of N specific antibodies, that are not induced in the vaccination. Spike specific antibody titers in this group were found substantially higher as compared to the only vaccinated group. A second vaccination in the previously infected group barely affected the antibody response, even in this oldest age group. Besides the limited number of participants, this information is important for guidance for the vaccination policies in the previously infected individuals. Also T cell responses were found enhanced in the previously infected group.Parry et al., examine anti-SARS-CoV-2 antibody and T cell responses following BNT162b2 vaccination in a cohort of 100 people aged 80-96 years. They showed that the majority of donors (98%) had spike-specific antibody responses but only 63% had detectable T cell responses against spike. These antibody and T cell responses did not correlate with age. Individuals who had prior infection had significantly higher antibody and T cell responses following vaccination. Finally, the authors showed that vaccinated individuals had strong neutralisation of a prototype strain of SARS-CoV-2, but reduced neutralisation against the P.1 variant of concern.Strengths:The authors have analysed antibody and T cell responses in a large group of people above the age of 80 (n=100), robustly showing that the elderly can respond to the Pfizer BNT162b2 vaccine and mount a strong antibody response. This data should instil confidence in the public that the elderly can mount a protective immune response against SARS-CoV-2 following vaccination with the Pfizer vaccine.The authors also demonstrate that the strength of antibody and T cell responses did not correlate negatively with age (at least within the ages of 80-96).Weaknesses:While the authors achieved their aim in examining whether elderly individuals could mount an immune response against SARS-CoV-2 following vaccination, this paper lacked a control group of vaccinated younger individuals to make some valuable and interesting comparisons. With a group of younger individuals who received the Pfizer vaccine, the authors would be able to directly compare whether the elderly had weaker antibody and T cell responses following vaccination.Opportunity:The authors could also have analysed neutralisation against other variants of concern . Essential revisions:1. The absence of a pre-vaccination sample makes the visualization of the vaccine response complicated. Moreover, visualization of a robust vaccine response and low pre-vaccination titers would strengthen the data. Why did the authors not include a pre-vaccination sample? This is also of importance with respect to point 3, the use of many different antibody testsThank you for your comment. We agree that it would have been useful to obtain a pre-vaccine sample but, as vaccination was approved rapidly in the UK, participants recruited to this study were some of the first in the world to receive the Pfizer-BioNTech vaccine outside of the registration trials. Vaccination centres were operating during a national lock down and study set up, ethical permission, and logistics for obtaining a phlebotomy sample  were such that we were unable to obtain an initial pre-vaccine sample. We do, of course, use nucleocapsid-specific antibody assessment to define ‘pre-infected’ donors but the great majority of donors were infection-naïve and it would be expected that spike-antibody levels would have been undetectable in this cohort.2. The absence of comparison with a younger age group limits the use of the data presented in the paper. Even though robust antibody and cellular responses are shown in this age group, we don't know how that related to younger age groups and how 'impaired' immune responses are in this old age group. Do the authors have the possibility to compare their data to a younger age group? The data on cross-neutralization against the Brazilian variant is highly interesting. How does this difference in neutralization between the strains compares to younger age groups?Thank you. It would have been interesting to directly compare against younger donors but in the UK the standard 3-week interval vaccine was used only donors aged >80 years and health care workers. This is because, after these initial programmes, there was a mandatory increase in the time between doses. HCW are not an ideal control for this cohort as rates of viral exposure are likely to differ from our elderly cohort who have been ‘shielding’.Salvagno et al., utilised the same assay platform as we describe and found, in health care workers with a median age of 44 years and without previous natural infection, a median titre of 1364 U/ml (761-2174) with 100% seropositivity following the second vaccine at day 50. This result is similar to our finding of 1138 U/ml following the second vaccine in those over 80. We have now added this to the list of references.We agree that the data for neutralisation of P.1 are interesting. In addition to reference 20, we have also added a new reference from Wang et al., who report a 3.8-4.8-fold decrease in live virus neutralisation of this variant in younger donors.3. The authors use many different antibody tests to analyze their data. How do these compare? What is the rational of using all these different tests? Are these tests standardized and validated to golden standards? Why do the authors use a different test for the measurement of the serum and DBS antibodies? This makes the comparison of serum to DBS complicated and less fair. The use of so many different tests is confusing, as are their read outs; do they all measure IgG, or what are differences? Please provide a rationale for this per figure and explain differences more clearly. This aspect really affects clarity of the paper and needs additional attention.Thank you. We agree that the study used several platforms and, whilst one advantage of this is that it allows relative comparison between assays, it is important to justify why this was done. There were several reasons for this approach.We utilised the Roche assay wherever a serum sample was available as this provided information on previous natural infection and also Roche is becoming an international standard test. The Roche assay provides IgG, A and M data against the receptor binding domain of Spike, rather than the total spike. This information has been added to the methods section. The Comparison data is available for Roche versus TBS DBS in figure 1C, with good correlation seen.The MSD assay was used as this allowed us to assess the difference in IgG antibody response against spike and the receptor binding domain of the spike protein that is known to be important for neutralisation. This data was not available from the other platforms.We were only able to obtain a pre-second vaccine sample by dried blood spot testing as phlebotomy bleed at this time point was not available as previously explained. The assay that has been developed for DBS testing is the Binding site assay which uses full trimeric spike and assesses the IgG, IgA and IgM response against Spike. The TBS DBS has been validated against WHO standard.These different assays will allow other groups to assess the correlation between assays for their studies. To improve the clarity of what each assay reports, the subtitle for each assay in the methods section has been modified to incorporate this information.4. The responses in the cellular tests are rather low with only 63% being positive. Which controls were included by the authors to investigate the robustness of the assay? These data are also rather limited by the lack of a pre-vaccination measurement. Do the authors have the possibility to compare to a pre-vac sample? As is done for the antibodies in Figure 1D. or to compare the results to a younger age group, to increase the reliability of the data shown.Thank you for your comment.The ELISpot assays were performed at Oxford Immunotec and is a standard assay that is widely used (https://www.tspotcovid.com) for diagnostic testing, vaccine assessment and research studies. As such we are confident in its performance although different cellular assays within laboratories will show variation in sensitivity.It was not feasible to get a blood sample for PBMC extraction and cellular work at the pre-vaccination time point for the reasons stated in the answer to question 1. In order to obtain some information at the pre-second vaccine time point, we utilised DBS for Ab responses as this was performed as a self-testing kit. Unfortunately, DBS is only useful for serological assays and does not permit any cellular work.Unfortunately we do not have a younger cohort who received 2 doses of Pfizer-BioNtech 3 weeks apart as this was not offered to younger ages in the UK and Oxford Immunotec do not hold any data for younger cohorts in this setting. However, we agree that the impact of immune senescence on cellular responses is a critical topic for future assessment.5. We feel that the correlation in Figure 1B is statistically wrong, due to the inclusion of so many negatives for nucleocapsid. We would suggest to analyze this differently, by just comparing the spike response in 2 groups: the N positive and N negative ones. A correlation can be made only in the N positive ones.Thank you. We have re-analysed the data as you suggest and now included an updated graph and correlation of those donors who are just nucleocapsid positive in Figure 1B. The correlation is indeed no longer present and we have changed the text accordingly.6. Figure 1D: donors with previous exposure and no-previous exposure could be separated into two graphs to more clearly show that antibodies in those with prior exposure do not increase much after the second dose.Thank you. We have separated the graphs and aligned them next to each other in figure 1D and 1E and the text has been amended accordingly.7. Line 267 – the authors mention that they examined cellular responses against spike, membrane and nucleocapsid but only the responses against spike are shown in Figure 2. Lines 274-275 – the cellular responses against N are only mentioned in text. These data should be shown as figures.Figure 2D and 2E have now been added to show the data for nucleocaspsid and membrane domain cellular responses in relation to previous natural infection and the text amended to incorporate these findings.8. Why is the timepoint 2 weeks following vaccination chosen, and not 28 days post-vaccination as done in many clinical trials? How do the authors suggest that the responses do compare?This time point was chosen to align with the original NEJM paper analysing the immunogenicity of the BNT162b2 vaccine from the phase 1 trial. Samples were taken at 7 and 14 days post the booster vaccine in this study which can be found at https://www.nejm.org/doi/full/10.1056/NEJMoa2027906. Based on the 30mg dose data in participants aged 65-85 years the geometric mean concentration of S1 binding IgG was 6014 U/ml whilst in our paper over those aged 80 and older, at the same time point was 1138 U/ml but this was utilising a different assay platform.9. How were the 20 donors down-selected for the neutralization assays in Figure 4? Was this randomized?Thank you. Capacity for live virus neutralisation is limited and it was not possible to assess the whole cohort. In order to include donors with a range of antibody responses participants were selected based on magnitude of Spike-specific response, 4 with the greatest response, 8 intermediate and 8 with low values. This has been added to the text.10. Figure 4: it would be interesting to determine whether neutralization against a more neutralization-resistant variant (B.1.351 or B.1.617.2) is maintained in the elderly.Yes, we agree. We are about to embark on further sampling of the whole cohort at a longitudinal time point (8 months since the booster vaccine) and intend to test the sera against variants of concern.11. The correlation between age and the humoral and cellular response is difficult to interpret. Given the restricted age range, it can be expected that there will be limited differences, unless there are major differences in health between these individuals. The title of this paragraph should be tuned down and the restriction of this analysis should be mentioned in the Discussion section.Thank you. We have changed the title of this section accordingly and have added to the Discussion regarding this limitation.12. The Discussion section is somewhat limited. The authors should at least discuss the limitations of the study and indicate what additional information would be needed to draw firm conclusions.Thank you for this advice. We have now added a paragraph to the Discussion to highlight the limitations of this study and also the requirement for longitudinal data on this cohort.“One of the limitations to this study includes the lack of pre-vaccination sample which is a reflection of the speed at which the vaccination programme was rolled out in people over 80 years old and the challenge of operating within vaccine centres during national ‘lockdown’. Future work should assess the longevity of the observed responses and neutralisation of new variants that have emerged since the vaccination programme started. This is now of great interest and may help to guide the need for further booster doses. Our work has focused solely on donors aged 80 years and older and, as such, it will also be important to see how immunity compares in younger cohorts who receive the BNT162b2 vaccine on a 3 week interval”.13. Please rephrase the sentences 74 to 80 in the introduction, which lacks proper understanding of immunological ageing.Thank you. We agree that this was a poorly written section and has now been replaced.14. What do the authors mean by independently-living people as mentioned in sentence 87 in the introduction? Is that the proper terminology? Is community dwelling more appropriate?Thank you and we apologise for confusion. We have now elaborated on this in the text. These were individuals in community dwellings that did not require any assistance in daily living or care and were able to attend a vaccination hub.15. Throughout the paper (including the title), the authors should avoid referring to the variants of concern with their country of origin. Either use the pango lineage names (P.1) or WHO's new Greek alphabet naming system for VOCs, in which P.1 is the γ variant.Thank you. We have edited the text to contain the pango lineage throughout. We have left the Victoria designation as this is not country of origin but refers to first sequence prototype.


In yeast and human cells many of the ribosomal proteins (r-proteins) are required for the stabilisation and productive processing of rRNA precursors. Functional coupling of r-protein assembly with the stabilisation and maturation of subunit precursors potentially promotes the production of ribosomes with defined composition. To further decipher mechanisms of such an intrinsic quality control pathway we analysed here the contribution of three yeast large ribosomal subunit r-proteins rpL2 (uL2), rpL25 (uL23) and rpL34 (eL34) for intermediate nuclear subunit folding steps. Structure models obtained from single particle cryo-electron microscopy analyses provided evidence for specific and hierarchic effects on the stable positioning and remodelling of large ribosomal subunit domains. Based on these structural and previous biochemical data we discuss possible mechanisms of r-protein dependent hierarchic domain arrangement and the resulting impact on the stability of misassembled subunits. S. cerevisiae  as in other eukaryotic model organisms most of these steps take place in the nucleus, thus requiring subsequent nucleo-cytoplasmic transport of the largely matured small and large ribosomal subunit  precursors  and protein (r-protein) components and their accurate assembly and folding. The rRNAs are initially synthesized as part of precursor transcripts (pre-rRNA) which are extensively processed and modified. In the yeast iewed in ). In yeaiewed in –6).The structure determination of a wide range of nuclear LSU precursor populations isolated from yeast wildtype or mutant strains indicated that the orientations of the seven major LSU rRNA secondary structure domains   processing and factor recruitment and release events is thought to be largely caused by hierarchical interrelationships between them. In support of this, the efficient nuclear export and the progression of many of the pre-rRNA processing steps depend on the ongoing r-protein assembly in specific ribosomal subregions . In caseFunctional coupling of r-protein assembly with the stabilisation and maturation of LSU precursors potentially serves to promote the production of ribosomes with defined composition. To further decipher mechanisms of such an intrinsic quality control pathway we analysed in the present work the impact of selected yeast LSU r-proteins on the progression of LSU precursor folding. Typically, r-proteins link several rRNA secondary structure elements in one or more rRNA secondary structure domains by direct physical contacts. These interactions have the potential to favour the establishment and stabilisation of the mature tertiary fold in individual RNA secondary structure domains  and to oThree yeast r-proteins rpL2 (uL2), rpL25 (uL23) and rpL34 (eL34) were included in the present study which are all required for the stabilisation and processing of intermediary nuclear LSU precursors ,24,25. IWe wondered if the three r-proteins rpL2, rpL25 and rpL34 with their different degrees of connectivity in LSUs have a global, a local, or any impact on the yeast pre-LSU folding pathway. And, if these effects might help to further explain their previously observed roles for LSU maturation and stability. To this end, LSU precursor particles were purified from yeast strains in which expression of one of the three r-proteins was shut down. In each case the structures of several particle populations could be resolved by single particle cryo-EM. Based on derived structure models we discuss the possible causes and the functional significance of the observed effects on the yeast LSU pre-rRNA folding pathway.To analyse changes in the folding states of intermediate nuclear LSU precursors upon lack of the r-proteins rpL2, rpL25 or rpL34, yeast strains were used which conditionally express one of these r-proteins under control of the GAL1/10 promoter ,25. In ain vivo depletion of rpL21  were observed in single particle cryo-EM analyses see . DefinedExpression shut down of LSU rRNA domain I and III binder rpL25 led to similar effects on the pre-LSU folding pathway. Again, two major folding states, Nog1TAP_L25-A and Nog1TAP_L25-B, were detected in Nog1-TAP samples purified from cells where expression of rpL25 was shut down see . The facIn summary these structural studies indicated that both rpL25 and rpL34 have a crucial role for the stable positioning of LSU rRNA domain III in nuclear LSU precursors. Moreover, they provided evidence that initial positioning and remodelling of LSU rRNA domains IV and V and the 5S RNP depend on the upstream assembly of rpL25 and rpL34.Further single molecule cryo-EM analyses revealed a different folding phenotype for Nog1-TAP associated LSU particles from cells in which expression of rpL2 was shut down. RpL2 is required for the trimming of the ITS2 spacer towards the 5.8S rRNA 3’ end. Hence, here LSU pre-rRNA processing is impaired just downstream of the rpL25 and rpL34 dependent initial cleavage inside the ITS2 spacer. As mentioned above, rpL2 is a multi-domain binder connecting at the subunit interface in a complex with rpL43 the LSU rRNA domains II, III, IV and V.in vivo depletion of rpL2  and the ITS2 binding factor Nop15 can be detected already before the LSU rRNA domain III in LSU precursor density maps rRNA signals were detected on the washed membranes using a Typhoon Imager FLA9500 . For re-probing, membranes were incubated twice with 100 millilitres of a boiling 0.1% (w/v) SDS solution in water. Each time the solution was let cool down to ambient temperature and then discarded. Signals were quantified using ImageJ.RNA extraction from samples taken during affinity purification was performed as described in  with minCryo-EM data processing was performed in Relion 3.0 . Beam inS. cerevisiae Has1 was taken from pdb 6c0f Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. 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: YesReviewer #2: YesReviewer #3: Partly**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/AReviewer #2: I Don't KnowReviewer #3: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?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.The Reviewer #1: YesReviewer #2: YesReviewer #3: 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: YesReviewer #2: YesReviewer #3: Yes**********5. Review Comments to the AuthorPlease 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. Reviewer #1: This is an excellent study analyzing structural changes of LSU precursors upon depletion of three selected large ribosomal subunit proteins. The manuscript is well written and the results add important new knowledge to the field. Still, some improvements are necessary to enhance the readability of the paper.- My main concern is the presentation of the Figures. None of the proteins or RNA domains visible in the structures are labeled. Therefore, it is difficult to follow the descriptions in the text. It cannot be expected that all readers are familiar enough with the structures to recognize all the elements described in the text without labels in the Figures.- Moreover, the names of the shown particel populations are counter-intuitive. The first population that is discussed is Nog1-TAP-F, which is shown in Figures 2A and 3A. Population E is shown in Figures 2B and 3B, etc. If the authors want to stick with this nomenclature, the names of the classes should also be indicated directly in the Figures.- Figures 2F and 3F are not mentioned in the text- Some kind of summary Figure in the end would be useful. E.g. an overview of the classes found in WT, with percentage of representation relative to the total number of particles for each class, and an indication which of these classes (or similar structures) are found upon depletion of the individual RPL proteins - The abstract is very general. The abstract would be more informative if at least the names of the three proteins whose role in subunit folding was studied were mentioned.- Figure Legend S1: the text for (B) is missingReviewer #2: Review for Manuscript ID PONE-D-21-15939 entitled " Analysis of subunit folding contribution of three yeast large ribosomal subunit proteins required for stabilisation and processing of intermediate nuclear rRNA precursors."Functional coupling of r-protein assembly with the stabilization and maturation of subunit precursors potentially promotes the cellular production of ribosomes. However, the precise mechanism remains exclusive. In this study, the authors tried to decipher an intrinsic quality control pathway by the evaluation of the contribution of three yeast large ribosomal subunit r-proteins, rpL2, rpL25, rpL34, for intermediate nuclear subunit folding steps. They found that a strong impact for rpL2, rpL25, and rpL34 assembly on hierarchical folding of several full LSU (sub-)domains that provide new insights into their role in subunit stabilization. In the rpL2 mutant strain, they observed folding deficiencies in LSU rRNA domains IV and V at the subunit interface and a premature 5S RNP orientation. The folding states of LSU precursors depleted of rpL2 provided direct evidence for its importance for the positioning of the domain IV and for the related LSU subunit interface remodeling. In the rpL25 and rpL34 mutant strains, they observed the 5S RNP could not be visualized and LSU rRNA domain III was not yet stably positioned in addition to LSU rRNA domains IV and V.Based on these observations, they propose that insufficient r-protein assembly might speed up degradation through the observed blockage at key points of the LSU folding pathway. Their proposal will be of broad interest and potentially interesting. However, appropriate revision is needed before publication in PLOS ONE. They give appropriate credits to previous work.Comments:1. The authors should demonstrate the Figures in a more appropriate way for general readers. More information related to three rpL proteins should be included. For instance, it is better to demonstrate the position of three rpL proteins in Figure 2. In addition, the superimposing of the WT and the rpL-depleted structures could clarify their differences.Reviewer #3: The authors of the paper entitle “Analysis of subunit folding contribution of three yeast large ribosomal subunit proteins required for stabilisation and processing of intermediate nuclear rRNA precursors” studied the effects of three r-proteins rpL2, rpL25 and rpL34 on the maturation of yeast ribosomes by analyzing post-transcriptional processing of ribosomal RNA and structures obtained by cryo-EM of several ribo-particles precursors of ribosome maturation. The authors discovered that the absence of expression of either the rpL25 or rpL34 affect the post-transcriptional cleavage of the internal spacer that connect the 5.8S rRNA with the 28S rRNA. These effects are interconnected with the absence of structuration of the 28S rRNA domain III. On the other hand, the absence of expression of the rpL2 protein affect trimming of the 7S RNA which is necessary to generate a mature 5.8S rRNA. Also, the absence of the rpL2 protein affect structuration of the domains IV and V as expected. Their results suggest possible steps in the maturation of eukaryotic ribosomes where the rpL34 and rpL25 are needed to structure domain III which will stablish maturation of the pre-rRNA and posterior interactions of the rpL2 protein required for further maturation of the newly formed 5.8S rRNA and domains IV and V.General observationsThis study had got very exciting structures that would complement previous work on ribosome maturation. However, this reviewer had hard time following the results’ descriptions with the provided figures, mostly because there is no enough information in the figures that can help the reader to follow the text of the document. Also, there are some questions about the results that this reviewer considers important to discuss.1) Supplementary Figure 1. This figure is important for the paper and very complex which legend do not have enough information for each of its panels. Unfortunately, the authors use the nomination D1-7 that is confusing with the rest of the paper. The authors should consider sticking with the usage of DI-DVII. Also, I am wondering if using the secondary structure of the 28S rRNA/5.8S rRNA would be better option to color code the interaction sites of the r-Proteins.2) What do the authors mean with “Production of ribosomes with defined composition”? Also, I do not see at the discussion section anything about this term.3) I could not find any reason of why the authors decided to use these three proteins and not others? The paper would benefit from the addition of a sentence of two explaining the reasoning of using these proteins, perhaps using as a reference supplementary figure 1.4) Similar to question 3, I could not find any reasoning on why the authors decided to use Nog1 as an anchor to pull out ribonucleoproteins? Also, supplementary figure 2 do not show such nog1-Tap growth delay as stated in the text. I am wondering if I am missing something. Size of the colony?5) On materials and methods. For how long the cultures grew under the presence of galactose? I agree that reduction of growth could be a good indication of the absence of the studied r-proteins. However, I am wondering if the cells are really lacking of these proteins in the selected period of growth. Did the authors performed western blots to follow the presence of the studied r-proteins after shifting the culture to glucose ? Also, I am wondering about the type of ribonucleoprotein materials that were present in the cells during the purification procedure. Did the authors analyzed polysome profilings before and after turning down the expression of the studied r-proteins? Perhaps they are losing information by pulling out just specific complexes attached to Nog1.6) About Figure 1. Consider to add densitometry calculations of each band to support your statements in the text. Also, why is 5.8S observed at the rpL34 and rpL25 samples if 7S was not produced? Is 5.8S very stable and perhaps coming from previous mature ribosomes? Why is that at the rpL2 sample the 25.5S signal is not as abundant as the control when the 7S is?7) Figures 2 and 3 needs extra labels. Please consider to indicate the location of the 5S rRNA, rpL2, rpL25, rpL34, and Nog1. Also, consider to mark the most important differences between structures.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  3 Sep 2021Dear Dr. Preiss,Thank you for taking the time to serve as scientific editor for our submitted manuscript.We are also very grateful to the three reviewers for carefully going through the manuscript and for their constructive reviews! We agree with the reviewers that any means to facilitate access of readers to the obtained twelve complex structure models besides the two-dimensional overviews in Figs 2-6 and the twelve interaction maps in S1 Appendix would be beneficial. To tackle this issue, we followed the advice of the reviewers by adding further labels for 5S rRNA, Nog1, the rpl2/rpl43 heterodimer and rpL25 in the reference structures of control samples shown in Figs 2-3. As suggested, the names of the structure models are now also directly shown in Figs 2-6.Secondly, we created in total five additional movies each showing animations of some of the structure models together with respective legends: The first one shows the positions of the three studied r-proteins with respect to the LSU rRNA domains in mature ribosomes (S1 Movie). The second one visualizes the hypothetical model-derived folding-path of rRNA domains during nuclear LSU maturation in control cells (S2 Movie). The next three movies show the observed folding states of immature ribosomal particles obtained from cells depleted of either rpL34, rpL25 or rpL2 . Structures shown in the S2-S5 Movies can be directly compared with each other. This should further facilitate tracing of the observed major differences independent of any structure model visualization software. For readers which want to directly analyze the structure models and electron density files these are hold for release until publication in the wwwpdb and the EMDB database. The respective accession codes are indicated in S3 Appendix. The original electron microscopy micrographs were uploaded on the EMPIAR database and can be accessed with the updated accession codes provided in S3 Appendix. Besides, we added as suggested by reviewer #3 the results of quantitation of the northern blots shown in Fig 1 as supplementary information S8 Appendix. In Fig 1 uncropped regions are shown with complete lanes of the northern blots developed with the indicated probes. We assume that requirements of PlosOne for blot and gel representations should thus be fulfilled.Finally, we updated the manuscript to include references to the newly added or changed Figures and we deleted as requested the funding information previously given in the Acknowledgement section of the manuscript.In the following we provide a detailed point by point response to the issues raised by the three reviewers.Reviewer #1- My main concern is the presentation of the Figures. None of the proteins or RNA domains visible in the structures are labeled. Therefore, it is difficult to follow the descriptions in the text. It cannot be expected that all readers are familiar enough with the structures to recognize all the elements described in the text without labels in the Figures.As explained above, we added labels for 5S rRNA, Nog1, the rpl2/rpl43 heterodimer and rpL25 to the reference structures in Figs 2-3 in addition to the previous color coding and labels according to the reviewer’s suggestions. This should further facilitate comprehension of the complicated structure models for readers not closely familiar with these ribosomal structures. In addition, we added five movies showing animations of the structure models together with respective legends to better visualize their three-dimensional organisation. Reference to these movies was added at the relevant locations in the main text.- Moreover, the names of the shown particel populations are counter-intuitive. The first population that is discussed is Nog1-TAP-F, which is shown in Figures 2A and 3A. Population E is shown in Figures 2B and 3B, etc. If the authors want to stick with this nomenclature, the names of the classes should also be indicated directly in the Figures.To avoid any confusion, we now show directly in Figs 2-6 the respective names of the structures as used in the text. - Figures 2F and 3F are not mentioned in the textReference to the mature ribosomal models in Figs 2 and 3 F was now added in the main text.- Some kind of summary Figure in the end would be useful. E.g. an overview of the classes found in WT, with percentage of representation relative to the total number of particles for each class, and an indication which of these classes (or similar structures) are found upon depletion of the individual RPL proteins We think that the newly added S2-5 Movies should provide a decent overview of folding states and their respective hypothetical transitions in the control cells (S2 Movie) and of residual folding states found in the mutant strains (S3-5 Movies). Camera perspectives, animations and legends were maintained in a way to allow for direct comparison of the structures shown in the movies. We indicated the percent of particles  used for calculation of densities of the respective particle population in the Legends of the new Figures. - The abstract is very general. The abstract would be more informative if at least the names of the three proteins whose role in subunit folding was studied were mentioned.As suggested, we added in the abstract the names of the three r-proteins studied in this work.- Figure Legend S1: the text for (B) is missingWe corrected the Figure Legend accordingly.Reviewer #2- The authors should demonstrate the Figures in a more appropriate way for general readers. More information related to three rpL proteins should be included. For instance, it is better to demonstrate the position of three rpL proteins in Figure 2. In addition, the superimposing of the WT and the rpL-depleted structures could clarify their differences.As explained in response to reviewer #1:“We added labels for 5S rRNA, Nog1, the rpl2/rpl43 heterodimer and rpL25 to the reference structures in Figs 2-3 in addition to the previous colour coding and labels according to the reviewer’s suggestions. This should further facilitate comprehension of the complicated structure models for readers not closely familiar with these ribosomal structures. In addition, we added five movies showing animations of the structure models together with respective legends to better visualize their three-dimensional organization. Reference to these movies was added at the relevant locations in the main text. Camera perspectives, animations and legends were maintained in a way to allow for direct comparison of the structures shown in the movies.”Reviewer #3- This study had got very exciting structures that would complement previous work on ribosome maturation. However, this reviewer had hard time following the results’ descriptions with the provided figures, mostly because there is no enough information in the figures that can help the reader to follow the text of the document.As explained in response to reviewer #1:“We added labels for 5S rRNA, Nog1, the rpl2/rpl43 heterodimer and rpL25 to the reference structures in Figs 2-3 in addition to the previous color coding and labels according to the reviewer’s suggestions. In addition, we added five movies showing animations of the structure models together with respective legends to better visualize their three-dimensional organization. Reference to these movies was added at the relevant locations in the main text. Camera perspectives, animations and legends were maintained in a way to allow for direct comparison of the structures shown in the movies.”- Supplementary Figure 1. This figure is important for the paper and very complex which legend do not have enough information for each of its panels. Unfortunately, the authors use the nomination D1-7 that is confusing with the rest of the paper. The authors should consider sticking with the usage of DI-DVII. Also, I am wondering if using the secondary structure of the 28S rRNA/5.8S rRNA would be better option to color code the interaction sites of the r-Proteins.It is true that we introduced here an inconsistency in the terminology for the LSU rRNA domains and we forgot to mention this in the Figure Legend of S1 Appendix. This inconsistency was introduced due to the space limitations we faced when trying to summarize the proteins, rRNA elements and their interactions in the twelve obtained highly complex structure models in as simple as possible two-dimensional maps with their general layout maintained for all models. In terms of space, Arabic numbering for LSU rRNA domains gave some advantage over the original roman one. For this reason, we still would like to keep the numbering, and we explicitly mention this point in the new version of the manuscript in the Figure Legends of S1 Appendix.Concerning the color coding, the bars of the small diagram-inserts in the protein boxes represent relative amounts of interactions with rRNA domains and are colored according to the color code used for the rRNA domain bar-diagram at the bottom of the Figures. - What do the authors mean with “Production of ribosomes with defined composition”? Also, I do not see at the discussion section anything about this term.We used the term “..promotes the production of ribosomes with defined composition” referring to the idea that many, most or even all newly made cytoplasmic ribosomes in yeast might share a common set of RNA’s  and r-proteins  in a defined ration (1:1:1…). We do not use exactly this term again in the discussion section of the manuscript. Still, we discuss there that the strong and hierarchical effects on rRNA folding upon lack of individual r-proteins might promote the degradation of incomplete particles and use in this context for example the term “compositional control mechanism” in the last paragraph of the discussion section. - I could not find any reason of why the authors decided to use these three proteins and not others? The paper would benefit from the addition of a sentence of two explaining the reasoning of using these proteins, perhaps using as a reference supplementary figure 1.We were originally interested in r-proteins which possibly affect yeast intermediate nuclear LSU folding steps, mainly because studies in recent years showed that these particle populations are structurally well resolved in single particle cryo-EM . We speculated that r-proteins with known effects on intermediate to late nuclear pre-rRNA processing steps might play a role for these folding steps . We decided then to choose among them for comparison three r-proteins with different rRNA-domain interconnectivity level , rRNA processing phenotypes  and assembly behaviour . The latter aspects are described in the fourth paragraph of the introduction. To express the comparative concept of this study more clearly, we added an appropriate sentence to the fourth paragraph of the introduction. In addition, we refer there now to the new S1 Movie file in which binding sites of the three studied r-proteins in mature ribosomes are visualized which allows to compare the way how they establish interactions between and in rRNA domains.- Similar to question 3, I could not find any reasoning on why the authors decided to use Nog1 as an anchor to pull out ribonucleoproteins? Also, supplementary figure 2 do not show such nog1-Tap growth delay as stated in the text. I am wondering if I am missing something. Size of the colony?We added to the first paragraph of the results a sentence in which we explain that Nog1 was previously found to be part of a wide range of intermediate nuclear LSU precursor populations and that its association with LSU precursors was independent of the ongoing expression of rpL2, rpL25 and rpL34. These were arguments for us to choose Nog1 as bait protein. Concerning the growth delay, yes, the smaller colony size reflects in this experiment the stated growth delay. We added this statement to second paragraph of the results part. - On materials and methods. For how long the cultures grew under the presence of galactose? I agree that reduction of growth could be a good indication of the absence of the studied r-proteins. However, I am wondering if the cells are really lacking of these proteins in the selected period of growth. Did the authors performed western blots to follow the presence of the studied r-proteins after shifting the culture to glucose ? Also, I am wondering about the type of ribonucleoprotein materials that were present in the cells during the purification procedure. Did the authors analyzed polysome profilings before and after turning down the expression of the studied r-proteins? Perhaps they are losing information by pulling out just specific complexes attached to Nog1.All expression mutant strains were continuously grown in galactose containing medium for their cultivation. We previously observed for most of the analysed yeast pGAL1/10 driven ribosomal protein gene mutants, including the ones studied here, strong pre-rRNA processing phenotypes starting at around 2 hours depletion time in glucose containing medium . As stated in Materials and Methods, expression shut down in this study was done by incubation for four hours in glucose containing medium. Large quantities of mature ribosomes (>150.000) are observed in logarithmic growing yeast cells and, in agreement with their long half-life, substantial amounts of them can still be detected in cells after these and longer depletion times . The early onset of maturation phenotypes is consistent with the stoichiometric need for most of the ribosomal proteins during ongoing rRNA synthesis. This high demand can apparently neither be sufficiently supplied by recycling of the still existing pool of “old” ribosomal proteins which is incorporated in the still existing “old” ribosomes, nor by the residual low expression of respective r-proteins under control of the glucose-repressed GAL1/10 promoter. Regarding the last point mentioned by reviewer #3, we agree with the possibility that we still might lack some information here by choosing Nog1 as bait for purification. Particles associated with Nog1 might be folded in a way not compatible with the SPA approach used in this work, and ribosomal sub-populations might be missing due to the choice of the bait. Anyhow, we believe that comparing the results from samples of control cells and the three mutant strains using the very same bait protein and experimental approach, provided strong evidence for the observed significant differences in the respective folding pathways. - About Figure 1. Consider to add densitometry calculations of each band to support your statements in the text. Also, why is 5.8S observed at the rpL34 and rpL25 samples if 7S was not produced? Is 5.8S very stable and perhaps coming from previous mature ribosomes? Why is that at the rpL2 sample the 25.5S signal is not as abundant as the control when the 7S is?We added the quantification of these data now as S8 Appendix and refer to it at the appropriate place in the first paragraph of the result section. Regarding the 5.8S rRNA: yes, we consider this as the background level of mature ribosomes which pre-existed before in vivo depletion of the respective r-proteins started. An alternative explanation is that maturation of pre-rRNAs is strongly delayed at these points but not fully blocked allowing for some production of Nog1-TAP associated particles with matured pre-rRNA over time.In regard to the rRNA composition of the rpl2 sample: In samples of control cells (as after depletion of rpL21) comparably high levels of 25.5+25S, but also of 5.8S were detected. We take this as indication for substantial amounts of Nog1-TAP associated particles in which the ITS2 spacer was cleaved at C2 and at least partially removed in both directions. After rpL2 depletion the 3’ trimming of 7S pre-rRNA seems to be inefficient leading to an increase in the 7S pre-rRNA to 5.8S rRNA ratio. Still, the 7S pre-rRNA signal seems to be lower in the rpL2 sample than the sum of the 5.8S rRNA and 7S pre-rRNA in the control sample. We would interpret this as an indication that the total population of Nog1 associated pre-rRNA with cleaved ITS2 is lower after rpL2 depletion than in control samples That would be reflected by the mentioned lower 25.5 + 25S rRNA in the rpL2 sample when compared to the control sample. A possible additional imbalance of (pre-)5.8S rRNA and (pre-) p25S rRNA after depletion of rpL2 might indicate that the latter is the initial target of degradation pathways  in this situation. This would not be surprising considering the binding site of rpL2 in ribosomes and its observed effects on the pre-rRNA folding pathway. This could be of possible interest in future experiments and we thank reviewer #3 for sharing his observation. - Figures 2 and 3 needs extra labels. Please consider to indicate the location of the 5S rRNA, rpL2, rpL25, rpL34, and Nog1. Also, consider to mark the most important differences between structures.As stated above, we now added according to reviewer’s #3 suggestions labels for 5S rRNA, Nog1, the rpL2/rpL43 heterodimer and rpL25 in Figs 2-3. Due to rpL34 being largely embedded in LSU rRNA domain III (see S1 Movie) this one is hard to label in these figures. As described above, we also introduced Movies S2-5 in the revised version of the manuscript which we believe to be helpful for comparing structures obtained from samples of control cells, including their transitions, with the ones from samples of the mutant strains. 6 Oct 2021PONE-D-21-15939R1Analysis of subunit folding contribution of three yeast large ribosomal subunit proteins required for stabilisation and processing of intermediate nuclear rRNA precursors.PLOS ONEDear Dr. Milkereit,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 address the residual comments by one of the reviewers.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 submit your revised manuscript by Nov 20 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at 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. 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If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.Additional Editor Comments (if provided):[Note: HTML markup is below. Please do not edit.]Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. 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 #3: All comments have been addressed**********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: YesReviewer #3: Yes**********3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #3: Yes**********4. Have the authors made all data underlying the findings in their manuscript fully available?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.The Reviewer #1: YesReviewer #3: Yes**********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: YesReviewer #3: Yes**********6. Review Comments to the AuthorPlease 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. Reviewer #1: The added labels as well as the movies have improved the manuscript. Still, many parts of the text remain difficult to follow as the Figures are still not sufficiently labelled.Here are some examples:"In state Nog1TAP-E  a group of four factors  was possibly released from the subunit. In contrast to state Nog1TAP-F, these could not anymore be detected bound to the rRNA expansion segment ES7 and LSU rRNA domains I and II at the subunit solvent surface."I tried to identify these factors in the respective regions, However, I was not able to find these proteins supposed to be missing in State E compared to state F. It would be helpful to have e.g.arrows in State E pointing at the positions where proteins have left, or alternatively arrows in State F indicating the proteins that are leaving during the transition from F to E."Otherwise, a hallmark of state Nog1TAP-E was the appearance of density unambiguously attributable to LSU rRNA domain III and many of the r-proteins associated with it. Among them were rpL25 and rpL34,…"While rpL25 is labelled in Nog1-TAP-E particles, rpL34 is not (it is only labelled in later particles), therefore it is difficult to follow the descriptions in the text."Several factors  previously associated with LSU rRNA domains I, II and III were not anymore observed and thus possibly released or with flexible orientation. Various other factors  and r-proteins  newly appeared in Nog1TAP-C in association with the emerging rRNA domains."Same as before – without labeling, it is difficult to follow this text.I'm aware that the main point of this paper is not the wild-type particles  but changes observed in the particles after ribosomal protein depletion. Still, I believe it is important to first understand the maturation steps of the wild-type particles (which is only possible with better labeling), in order to be able to better compare to the alterations in ribosomal protein-depleted particles.Reviewer #3: (No Response)**********what does this mean?). If published, this will include your full peer review and any attached files.7. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  12 Oct 2021Dear Dr. Preiss, dear reviewers,We thank you again for taking the time to inspect the manuscript and for the constructive suggestions to improve its quality.www.pdb.org), the 2D-Maps in S1 Appendix were thought to enable an easier access to the 3D-structure data by showing for each of the twelve models the protein composition, the modeled RNA helices and a simplified 2D-interaction network between them. To follow, as suggested by reviewer 1, the changes in protein compositions also in 3D-representations of the structure models we created for the new revised version an additional supplementary Figure (S9 Appendix). Here, the structure models are visualized in 14 snapshots each with optimized orientation, coloring and zoom level to highlight specific observations raised in the text. We changed the manuscript accordingly, in which we refer now to this additional Figure at several points. We refer now also at a few more points to S1 Appendix with its 2D summary of the structure models. In addition, we corrected a wrong reference on page 5 (line 254) to 5S rRNA helices 5 and 6, now referring to 5S rRNA helices 4 and 5. Reviewer 1 raises the point that the labelling of the figures could still be improved (see below). We agree with reviewer 1 that parts of the text describing changes in the protein composition between different pre-ribosomal populations cannot be easily followed by using the visualization of the structure models shown in Figures 2-6. Due to the large number (twelve) of the described complex models these provide primarily an overview on the general observed rRNA fold from two defined orientations. It is often very challenging to point here on one specific protein out of the easily more than 40 protein components, with many of them embedded into the RNA and intertwined with each other. Besides providing the readers with all pdb models and density maps  a group of four factors  was possibly released from the subunit. In contrast to state Nog1TAP-F, these could not anymore be detected bound to the rRNA expansion segment ES7 and LSU rRNA domains I and II at the subunit solvent surface."I tried to identify these factors in the respective regions, However, I was not able to find these proteins supposed to be missing in State E compared to state F. It would be helpful to have e.g.arrows in State E pointing at the positions where proteins have left, or alternatively arrows in State F indicating the proteins that are leaving during the transition from F to E."Otherwise, a hallmark of state Nog1TAP-E was the appearance of density unambiguously attributable to LSU rRNA domain III and many of the r-proteins associated with it. Among them were rpL25 and rpL34,…"While rpL25 is labelled in Nog1-TAP-E particles, rpL34 is not (it is only labelled in later particles), therefore it is difficult to follow the descriptions in the text."Several factors  previously associated with LSU rRNA domains I, II and III were not anymore observed and thus possibly released or with flexible orientation. Various other factors  and r-proteins  newly appeared in Nog1TAP-C in association with the emerging rRNA domains."Same as before – without labeling, it is difficult to follow this text.I'm aware that the main point of this paper is not the wild-type particles  but changes observed in the particles after ribosomal protein depletion. Still, I believe it is important to first understand the maturation steps of the wild-type particles (which is only possible with better labeling), in order to be able to better compare to the alterations in ribosomal protein-depleted particles.”We thank reviewer 1 for his comments on the revised version of the manuscript. As described above, to provide a clean way to follow the changes in protein compositions in wildtype cells by 3D structure model representations we added now supplementary Appendix 9 to the manuscript. Here, 14 snapshots of the structure models are shown with optimized orientation, zoom level and specific coloring of protein groups of interest to visualize all the above mentioned and several other observations described in the text.AttachmentResponseToReviewers.docxSubmitted filename: Click here for additional data file. 18 Oct 2021Analysis of subunit folding contribution of three yeast large ribosomal subunit proteins required for stabilisation and processing of intermediate nuclear rRNA precursors.PONE-D-21-15939R2Dear Dr. Milkereit,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.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Thomas Preiss, PhDAcademic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments: 15 Nov 2021PONE-D-21-15939R2 Analysis of subunit folding contribution of three yeast large ribosomal subunit proteins required for stabilisation and processing of intermediate nuclear rRNA precursors. Dear Dr. Milkereit: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. onepress@plos.org.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 plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staffon behalf ofProf Thomas Preiss Academic EditorPLOS ONE