Antibody- and T Cell-Dependent Responses Elicited by a SARS-CoV-2 Adenoviral-Based Vaccine in a Socially Vulnerable Cohort of Elderly Individuals

Round 1

Reviewer 1 Report

Moya et a. report detection of antibodies (IgG) and T cell-dependent response in a cohort of patients with a mean age of ~72 years old.

Overall, the study and the data are presented with only limited clarity and detail/analysis; some results are mentioned in the Discussion, but no details (Methods and Results) are included. In particular, some ethical considerations should be clarified in order to leave no doubts that the study was conducted according to the Helsinki Declaration.

Major points

Ethical considerations. Lines 197-199 are concerning: “the County Government of the city of Cordoba (Argentina) ordered that the elderly residents of the long-term care facility will receive the Sputnik-V vaccine”. This choice of words would suggest that no informed consent was obtained. However, because lines 283 states “participants had to consent to participate” and lines 72-74 state that enrolled individuals “provided written informed consent before undergoing any study procedures”, it is reasonable to expect that informed consent was indeed obtained. However, the authors should avoid any potential misinterpretation and modify lines 197-199.

Lines 74-78. The participants were tested for IgG/IgM with a rapid test but it is not clear when, although it seems to reasonable to assume that this was done at the time of first and second dose? The Authors should clarify this. Lines 74-78 seem to suggest participants were tested for (ongoing?) asymptomatic infection with an IgG/IgM rapid test. The test’s manufacture only supports the use of its antibodies rapid test to detect a previous infection, not an ongoing one, as the test relies on detection of antibodies not viral antigens  (link to Abbot layman statements: https://content.veeabb.com/1d09429b-8373-419f-8f1a-d28f9586863a/66fb848d-ce53-4a71-9d89-5eafb1df1f30/66fb848d-ce53-4a71-9d89-5eafb1df1f30_source__v.pdf ). Overall, lines 74-78 are confusing and contradictory: were the participant tested or not tested for past infections? Lines 200-201 state that only participant with no previous exposure to SARS-CoV-2 were included, but asymptomatic infections are mentioned and, allegedly, grouped with those who have “not been infected” (Fig 2 legend). The Authors should clarify this important point and, if an IgG/IgM test was indeed performed when dose 1 was administered, the IgG data (Fig 1 etc) should be stratified to reflect this. Additionally, the Authors should note that N antibodies (IgG) decrease quickly after natural infection and therefore there is only a limited time window for N antobodies detection to be used to discriminate between vaccine vs natural infection (i.e. Fenwick t al., 2021 etc).

Trial exclusion criteria. The Authors state that participants were “healthy” (line 73), but also mention that the cohort of interest (elderly socially-vulnerable individuals) might contain immunocompromised individuals. How many participants were assessed and were some excluded because immunocompromised? If so, how is the final cohort representative of "a socially vulnerable cohort of elderly individuals”. Additionally, lines 147-148 state that 31.5% of the participants were “receiving palliative care” (or were semi-dependent, dependent). How was palliative care assessed in the context of decision to administer vaccine doses and discuss informed consent?

Methodology. For the assessment of T cell response, PBMC were obtained from EDTA bloods and then frozen. After resuscitation, cells were challenged with relevant peptides and Intracellular staining and flow cytometry were used to detect cytokines expression. The Authors should clarify why direct measurement of the produced-and-excreted cytokines (ELISA or bead-based flow cytometry) was not performed and reference how the method used (intracellular expression detected by flow cytometry) correlates to secreted level. Additionally, for flow cytometry (Fig 4), Fluorescence intensity of the Mean should be included alongside % of positive events and could be used to uncover differences in samples, i.e. stratified for age and/or pre-existing medical conditions, etc.

Methodology. One or two vaccine doses? In “Trial Procedure” it is stated that the vaccine was administered on days 1 and 21 of the study. In “Statistical Analysis” it is mentioned that “participants who had received at least one dose of two adenovirus vectors”. The Authors should clarify if/how many participants were excluded to reach the 72 adults mentioned in Trial Populations (Results; line 140).  

Fig 1 and Fig 2. Please, clarify how “asymptomatic infections” are grouped (with Infection?)

219-200 no data is shown to support this point. Please, either include the data or remove the paragraph.

Fig 3. A histogram would be more appropriate, with antibodies levels at the different time points grouped per age bracket. 

Fig 4. bottom panel: age stratification would be informative and add clarity, relevance and impact overall.

161-164: no data is shown and described (which viruses were tested? Was HBV the only one detected?). Please, either include the data or remove the paragraph.

219-223: data discussed but not included in Methods nor Results. Please, either include the data or remove the paragraph.

Minor Points:

Punctuation and spelling should be checked (i.e. line 69; line 109; line 114; line 219; etc)

Author Response

Mayor notes:

1) On February 2, 2021, the County Government of the city of Cordoba (Argentina) suggested that the elderly residents of the long-term care facility (Padre Lamónaca) will receive the Sputnik-V vaccine.

The word "ordered" was changed to the word "suggested", which implies the request of the consent of adults involved in the study.

2) We did not screen for evidence of past or current SARS-CoV-2 infection by testing blood or nasal specimens before enrollment. In other words, we did not test for antibodies or evidence of infection on the same day that the first dose of vaccine was given (baseline), but rather 21 days after the first dose was given, which coincides with the second dose of vaccine. However, in order to overcome this limitation, we determined whether individuals had asymptomatic infection prior to Sputnik-V vaccine administration, from the detection of the rapid test for SARS-CoV-2 nucleoprotein (Abbott Panbio, COVID-19 IgG/IgM). With the understanding that N antibodies (IgG) decrease rapidly after natural infection and therefore there is only a limited time window for N antibodies to detect past illnesses.

Figure 1. Efficacy of the Sputnik vaccine as measured by the percentage of individuals displaying RBD-specific IgG responses at days 21, 42 and 180 post-immunizations. These efficacy data were taken only in older adults who had not been previously infected with COVID-19, as detected by SARS-CoV-2 nucleoprotein.

This legend was added to figure 1.

3) The word "healthy" was change for the word "wholesome"

We proposed that there could be immunocompromised patients, as an assumption, because we dealing with a vulnerable population. No studies were carried out to determine the immune status of these patients. Therefore, no patient was excluded because of any type of immune compromise.

Of the total number of patients, 68.5% were self-supporting or independent and 31.5% were semi-dependent or dependent.

We apologize for a possible misunderstanding in the way we expressed it, so the sentence was changed as you can see above.

4) We thank the reviewer for this observation. For the assessment of T cells response, we used intracellular cytokine staining (ICS) upon antigen-specific stimulation of PBMCs. ICS is an easy, rapid and semiquantitative method that provides information of cytokine production/expression at individual cell level, allowing us to analyze the percentage of a specific cell population that produces a cytokine of interest. For instance, in heterogeneous populations, using ICS we can detect which source of CD8⁺ T cells produce TNF-α upon stimulation with SARS-CoV-2 specific peptide pools. On the other hand, ELISA or CBA is used to measure the total amount of secreted cytokines produced by all the cells in the culture and does not provide information on cytokine production at the single cell level. ICS is a well-known method widely used in literature for the detection of antigen-specific T cell response in different viral infections, specifically SARS-CoV-2.

We thank the reviewer for this suggestion. We agree with him/her comment about stratifying patients by age or pre-existing medical conditions.  Regarding the fluorescence intensity of the mean (MFI), we consider that the measurement of MFI to study differences among samples may not be suitable due to the small number of events included in the desired population (e.g., CD8⁺ positive for the cytokine of interest) for which the MFI value is calculated. The fact that a few events will be consider in MFI may be a source of error leading to overestimated or subestimated results.  For this reason, we decided to analyze our results by studying the percentage of events that produce the cytokine of interest, showing that this event (T cell) is specfically responding to SARS-CoV-2 stimulation. 

5) Safety analyses included all the participants who had received two doses of two adenovirus vectors (Ad26 – Ad5) vaccine. 

Here again there was a typo, all 72 older adults received the 2 doses of Sputnik-V vaccine.

6) Figure 2. Difference in RBD-specific IgG antibodies in potentially previously infected individuals based on SARS-CoV-2 nucleoprotein detection who were immunized with Sputnik vaccine versus those who were negative for SARS-CoV-2 nucleoprotein, taken as previously uninfected. 

The legends in Figures 1 and 2 were modified.

The paragraph in line 219 was removed.

7) The previous graph was changed to a bar graph, as suggested.

8) Likewise, exposure to other viruses, such as hepatitis B (HBV; core positive), hepatitis C and acquired immunodeficiency virus, was also reflected by an increase in the production of antibodies to SARS CoV-2 (9487.6 vs. 1832.2; p = 0.013), when compared to vaccinated individuals without previous infection. 

The other 2 viruses that were analyzed were added.

9) The data in paragraphs 219 - 223 have been removed from the text

Minor points

The changes suggested as minor points were made.

Reviewer 2 Report

The authors have explored the long-term effects of combined adenoviral-based vaccines in a socially-vulnerable elderly population in Cordoba, Argentina. The longitudinal sero-epidemiological study analyzed both humoral response (SARS-CoV-2-specific IgG for Spike RBD) and celluar response (SARS-CoV-2-specific T cell-mediated immunity). The results are interesting in that CD8-positive T cell responses increased despite the decreased humoral response, which have implications for long-term protection in socially vulnerable elderly individuals. But the following points raises concerns.

1. Low-income is a very elusive term. It may be specified in a scientific paper.
2. In Figure 2, it is not clear where [which comparison(s)] the p value (p=0.53) indicates. Also, the following in the parenthesis must be addressed. (Anti-SARS-CoV-2IgG -> Anti-SARS-CoV-2 IgG, Sputnik vaccine -> Sputnik V vaccine or Sputnik-V vaccine, asymptomatic -> asymptomatic.)
3. In Figure 3. the graph can be changed to include individual data based on age. Change the X-axis as the variable of age so that one can see the levels of RBD-specific IgG levels of particular age. For example, one cannot expect such huge change in IgG levels between age 69 and 70.
4. Sputnik vaccine may better be spelled out (Sputnik V vaccine or Sputnik-V vaccine) and used consistently throughout the manuscript. It may be the same with CD4 and CD8 T cells (CD4 and CD8 T cells -> CD4+ and CD8+ T cells)
5. In Figure 4, it is not clear what the graph means. Please explain in more detail what each axis (X and Y) means. Also, the meaning of unstimulated and stimulated must be explained in the legend.
6. This reviewer finds a lot of sloppiness in the manuscript. Many typos or grammatical errors must be addressed. Some of them are listed below with suggestions.

line 31: evidence -> evidenced

line 56: )

line 69: post-vaccination -> post-vaccination.

line 90: 0.5-ml -> 0.5 ml

line 109: (Abbott Panbio, COVID-19 IgG/IgM). test

line 114: DNasa -> DNase

line 118: added to cultures -> added to cultures

line 124: performed -> performed.

line 151: Spike RBD CMIA -> Spike RBD, CMIA

line 175: CD4 and CD8 T cells -> CD4+ and CD8+ T cells

line 176: reported control -> reported to control

line 205: offered, 90% protection -> offered 90% protection

line 219: homeless -> homeless.

line 233: .in -> in

line 267: at bay resistant -> at bay. Resistant

In Figures 2 & 4, Period(.) must be added at the end of the setence.

Author Response

1) The word low-income was changed to underprivileged population.

2) The legend in Figure 2 was changed to make the analysis more explicit.

3) 3) Graph 3 was changed to best represents the data.

4) The word Sputnik-V vaccine replaced all the existing names for the vaccine in the text.

The words CD4+ and CD8+ replaced all other words refering to CD4 and CD8 cells.

5) Figure 4 was changed, as well as its legend, to improve its understanding. 

6) The manuscript has been revised to avoid sloppiness. Thank you for pointing this out.