key: cord-1044868-5p7b28lj
authors: Sajadi, Mohammad M.; Myers, Amber; Logue, James; Saadat, Saman; Shokatpour, Narjes; Quinn, James; Newman, Michelle; Deming, Megan; Tehrani, Zahra Rikhtegaran; Karimi, Maryam; Abbasi, Abdolrahim; Shlyak, Mike; Frieman, Matthew B.; Crotty, Shane; Harris, Anthony D.
title: Mucosal and systemic responses to SARS-CoV-2 vaccination in infection naïve and experienced individuals
date: 2021-12-14
journal: bioRxiv
DOI: 10.1101/2021.12.13.472159
sha: 217e1803ff5d8129c122d395959d2d0385602a73
doc_id: 1044868
cord_uid: 5p7b28lj

With much of the world infected with or vaccinated against SARS-CoV-2, understanding the immune responses to the SARS-CoV-2 spike (S) protein in different situations is crucial to controlling the pandemic. We studied the clinical, systemic, mucosal, and cellular responses to two doses of SARS-CoV-2 mRNA vaccines in 62 individuals with and without prior SARS-CoV-2 exposure that were divided into three groups based on serostatus and/or degree of symptoms: Antibody negative, Asymptomatic, and Symptomatic. In the previously SARS-CoV-2-infected (SARS2-infected) Asymptomatic and Symptomatic groups, symptoms related to a recall response were elicited after the first vaccination. Anti-S trimer IgA and IgG levels peaked after 1st vaccination in the SARS2-infected groups, and were higher that the in the SARS2-naive group in the plasma and nasal samples at all time points. Neutralizing antibodies titers were also higher against the WA-1 and B.1.617.2 (Delta) variants of SARS-CoV-2 in the SARS2-infected compared to SARS2-naïve vaccinees. After the first vaccination, differences in cellular immunity were not evident between groups, but the AIM+ CD4+ cell response correlated with durability of humoral immunity against the SARS-CoV-2 S protein. In those SARS2-infected, the number of vaccinations needed for protection, the durability, and need for boosters are unknown. However, the lingering differences between the SARS2-infected and SARS2-naïve up to 10 months post-vaccination could explain the decreased reinfection rates in the SARS2-infected vaccinees recently reported and suggests that additional strategies (such as boosting of the SARS2-naïve vaccinees) are needed to narrow the differences observed between these groups.

Although several recent studies have shown protection from reinfection amongst those with SARS-CoV-2 infection who are subsequently vaccinated (1, 2) , controversy exists as to the degree of protection from reinfection in those with prior COVID-19 infection, as well as the role for vaccination and boosters in this population. Understanding the primary immune responses to natural infection and vaccination, as well as the secondary responses in those previously infected or vaccinated is key to understanding the protection.

Given the recent findings of a decrease in durability of protection after vaccination (3) , this has become even more urgent. The current study was undertaken to investigate the primary responses in COVID-19 naïve vaccinees, as well as secondary responses in those with previous COVID-19. To our knowledge, no group has comprehensively studied these responses to include clinical symptoms, circulating binding and neutralization, and cellular and mucosal immunity.

A total of 3816 health care workers (HCW) were enrolled in the serosurvey study (4) , of which 151 were randomly contacted, and 67 volunteers enrolled. Of the 67, 62 had received both vaccine doses (4 had dropped out prior to second vaccine and one was diagnosed with COVID-19 before second vaccine). The 62 who received both vaccines divided into the following groups: 19 Antibody negative, 17

Asymptomatic antibody positive, and 26 Symptomatic antibody positive (Table 1) . After the first vaccination, individuals in the Asymptomatic and Symptomatic groups had higher rates of systemic symptoms compared to those in the Antibody negative group, for fever, chills, fatigue, myalgia, headache in the Symptomatic group (Fig. 1) . After the second vaccination, the Ab negative had an increase in symptom reporting compared to the first vaccination, and any differences with the Symptomatic group were no longer statistically significant. However, after the second vaccination, the Asymptomatic Group had less fatigue, myalgias, and headaches compared to the Ab negative group (Fig.   1 ). After the first and second vaccination, the Asymptomatic group had less symptom reporting in nearly every category, and although the individual categories did not reach statistical significance, the Asymptomatic group had less systemic symptoms than the Symptomatic when considering the number of systemic symptoms reported in each category of 1 more (71% vs 96%), or 2 or more (41% vs 77%) systemic symptoms (P=.03 and P=.03, respectively). 

At baseline, the Antibody negative groups had undetectable endpoint titers of IgM, IgA, and IgG antibodies to the SARS-CoV-2 spike trimer, whereas these could be detected to varying degrees in those in the Asymptomatic and Symptomatic groups ( Fig. 2A) . IgM, IgA, and IgG S trimer endpoint titers continued to increase after 2 nd vaccination in the Ab negative group, while they plateaued after the first Ab negative, Asymptomatic, and Symptomatic groups shown in orange, blue, and white columns, respectively. Each symptom was assessed by 2-tailed Fisher's exact test between the Ab negative and the Asymptomatic or Symptomatic groups, with P values <.05 being considered significant and shown with asterisks above the columns.

A B vaccination in the Asymptomatic and Symptomatic Groups ( Fig. 2A) . IgG S trimer endpoint titers after each vaccination were higher in the Asymptomatic and Symptomatic Groups compared to the Ab negative group ( Fig. 2A) , and this difference continued until the study completion, up to 10 months after the second vaccination (Fig. 2B) . At the last time point the median endpoint titers for the Antibody negative group was 2,700, and statistically lower compared to the Asymptomatic (8,100) and

Symptomatic ( S1A) . The plasma samples were tested against the WA-1 and B.1.617.2 (Delta) variants of SARS-CoV-2. In the Ab negative group the neutralization titers for both Wuhan and Delta variants sequentially increased until 14 days after the second vaccination ( Fig. 4A and B) . In the Asymptomatic and Symptomatic groups, the endpoint titers peaked at 14 days after the first vaccination, and remained stable before and after the second vaccination. By 14 days after the second vaccination, median ID99 WA.1 neutralization titers were 5,120 in the Ab negative group, compared to the 51,200 in the Asymptomatic and 81,920 in the Symptomatic groups (P<.0001 for each) (Fig. 4A) . Comparing Moderna versus Pfizer recipients within 

We compared the CD4 + and CD8 + T cell reactivity of the three cohorts against SARS-CoV-2 S by activation induced cell marker (AIM) and intracellular cytokine staining (ICS) assays ( Fig. 5A , C, E, H, and figs. S3 and S4). When comparing the AIM responses at baseline, the Symptomatic group had significantly higher levels of CD4 and cTFH responses to Spike compared to the Antibody negative group (P<.0001 and P=.009, respectively) ( Fig. 5B and D) . The antibody negative group saw increases in CD4 and cTFH AIM responses after each vaccination, while AIM responses in the Asymptomatic and Symptomatic groups peaked after the first vaccination ( Fig. 5B and D) . There was no statistically significant difference between the AIM responses to CD4 or cTFH between the SARS2-naive and infected groups after the 1 st or 2 nd vaccinations ( Fig. 5B and D) .

In the CD4 + ICS assay, significantly lower S-specific IFNγ and cytokine staining were seen in the Antibody negative group at baseline compared to the Asymptomatic (P=.04 and P =.01, respectively) and

Symptomatic (P=.003 and P=.001, respectively) groups ( Fig. 5F-G) . Compared to baseline levels, after S-specific CD8 + T cell IFNγ and cytokine expression differences were noted between the Symptomatic and Antibody negative groups at baseline (P=.006 and P=.002, respectively) ( Fig. 5I and J) . Compared to baseline levels, after 1 st vaccination peak percentages of IFNγ and cytokine secreting cells significantly increase in the Antibody negative group only (P=.003 and P=.001, respectively). (Fig. 5I and J) . The percentage of IFNγ and cytokine secreting cells in the Asymptomatic and Symptomatic groups did not significantly change before or after vaccinations ( Fig. 5I and J) . There was a strong positive correlation between the AIM + CD4 + cells and percentage of IFNγ secreting CD4 + cells in each of the Antibody negative (P< .0001), Asymptomatic (P=.003), and Symptomatic (P< .0001) groups ( fig S5) . In the Antibody negative group, Moderna vaccines recipients had statistically significant higher S-specific CD4 + T cell and cTFH cells detected by AIM assay, and higher S-specific CD4 + T cell and CD8 + T cell ICS responses than Pfizer recipients 14 days after the second vaccine (figs S6 and S7).

The cellular and antibody data were analyzed for correlations. In the SARS2-infected groups, there were positive correlations between S-specific CD4 + cells (Aim + ) with S IgG titers pre-vaccination (P=.01), at The primary and secondary responses had a clear set of differences including symptoms and antibody levels.

After the first dose of vaccine, those with a history of SARS2 infection had more symptoms, consistent with a secondary immune response. Interestingly, those in the Symptomatic group also had a higher percentage of symptoms compared to the Asymptomatic group. Given the lack of significant immune differences observed between the two groups, this opens the possibility that the elevated secondary response to vaccination could be related to exaggerated response to antigens in this group (genetic differences), though it can also be a reflection of a higher viral burden during SARS2-infection (consequence of having more severe COVID-19). however, it remains unknown if 2 doses rather than 1 dose of an mRNA vaccine is needed for protection.

Importantly, one of these studies has shown that SARS2-infected vaccinees appear to have less reinfection than SARS2-naïve vaccinees (2) , and the finding of elevated binding and neutralization titers in the former group could explain the increased protection.

This study was continued 10 months after the second vaccination, so there was an opportunity to study the early durability of the responses in the different groups. There was no difference in the slope of decline between the groups, but the IgG antibody levels in the SARS2-infected groups remained higher at each time point, including at last time point. Interestingly, the AIM+ CD4 + cell response at baseline in the SARS2-infected correlated with IgG anti-S antibody responses, but only at baseline, 3 months, and 9-10 months. Given the lack of correlation with peak titers, this suggests that AIM + CD4 + response tracks with the durability rather than the magnitude of the IgG response. Neutralizing antibody also correlated with The strengths of this study include using a clearly defined cohort with regimented sampling points.

Additional strengths include use of vaccinees who received Moderna or Pfizer, as well as studying clinical, cellular, and mucosal parameters. This study was limited by sample size, and thus a larger sample size could have found additional differences between the groups based on prior SARS-CoV-2 exposure, type of vaccination, or other factors. Finally, although the studies undertaken were fairly comprehensive, some key sites involved in immune regulation (lymph nodes, lungs) were not involved in the current study.

This study found key differences in the response to SARS-CoV-2 vaccination depending on previous exposure to the virus. SARS2-infected individuals had higher binding and neutralization titers throughout the study. This was also true of the relevant classes of anti-S antibody (IgG and IgA) at mucosal sites which are likely involved in the protective efficacy of the mRNA vaccines. Finally, while differences in cellular immunity were less evident between groups after vaccination, there was data supporting that the AIM+ CD4 + cell response could be tied to durability of humoral immunity against the SARS-CoV-2 S protein.

In those with prior SARS2 infection, the number of vaccinations needed for protection, the durability, and need for boosters are unknown; however, the lingering differences between the SARS2infected and SARS2-naïve up to 10 months post-vaccination suggests that additional strategies (such as boosting of the SARS2-naïve vaccinees) are needed to narrow the immunological differences observed between the groups in this study, which are likely related to the

HCW who had previously enrolled in a hospital-wide serosurvey study in the summer of 2020 (4), conducted at the University of Maryland Medical Center, were randomly contacted based on stratification into three groups, as previously described (13) 

Live virus neutralization assays were carried out, as previously described (13, 25 

To quantify the S-specific T cell response, a S megapool (MP) containing overlapping peptides of the entire S protein sequence was used. In addition, MP remainder (MP_R) containing non-S epitopes in SARS-CoV-2 was used, as previously described (26) . Peripheral blood mononuclear cells (PBMCs) were incubated with 1 µg/mL of S MP or MP_R for 24 hours in a 96-well U bottom plate at 1x106 cells/well in RPMI media containing 5% human serum AB (Gemini Bio), 1% GlutaMAX (Thermo Fisher Scientific) and 1% Penicillin-Streptomycin (Thermo Fisher Scientific). DMSO (0.1%) and staphylococcal enterotoxin B (SEB, 1 µg/mL) were used as negative and positive controls, respectively. Fifteen minutes prior to the addition of the MPs, 0.5 µg/mL anti-CD40 mAb (Miltenyi Biotec) and CXCR5 antibody were added. Following the 24 hour incubation, PBMCs were surface stained with antibodies in Table S1 for 30 mins at 4°C, washed in PBS, fixed for 10 mins in 4% formaldehyde (BD Biosciences Cytofix), washed in PBS and acquired on a BD FACSCelesta Cell Analyzer. AIM+ antigen-specific CD4+ T cells were defined as (surface CD40L+ OX40+) and antigen-specific circulating T follicular helper (cTFH) cells were defined as (CXCR5+ surface CD40L+ OX40+).

PBMCs were incubated with 1 µg/mL of S MP or MP_R for 24 hours in a 96-well U bottom plate at 1x10 6 cells/well in RPMI media containing 5% human serum AB (Gemini Bio), 1% GlutaMAX (Thermo Fisher Scientific) and 1% Penicillin-Streptomycin (Thermo Fisher Scientific), as previously described (27) . DMSO (0.1%) and staphylococcal enterotoxin B (SEB, 1 µg/mL) were used as negative and positive controls, respectively. Fifteen minutes prior to the addition of the MPs, 0.5 µg/mL anti-CD40 mAb (Miltenyi Biotec) and CXCR5 antibody were added. Following the 24 hour incubation, monensin (BD Biosciences GolgiStop) and brefeldin A (BD Biosciences GolgiPlug) were added and PBMCs were incubated for an additional 4 hours. PBMCs were surface stained with antibodies in Table S2 for 30 mins at 4°C, washed in PBS, and then fixed and permeabilized (1x BD Biosciences Cytofix/Cytoperm) for 30 mins. After fixation, PBMCs were washed in 1x Perm/Wash buffer (BD Biosciences) and stained for 45 mins at 4°C with the intracellular antibody panel in Table S3 and acquired on a BD FACSCelesta Cell

Analyzer. Antigen-specific CD4+ T cells were defined as CD40L+ and IFNγ+, TNFα+, or IL-2+ and antigen-specific CD8+ T cells were defined as CD69+ and IFNγ+, TNFα+, or IL-2+.

The reciprocal end-point binding titers represent the maximal dilution of plasma that achieves binding.

For T cell assays, sample quality was assessed by CD4+ AIM+ (CD40L+ OX40+) SEB response.

Samples with a SEB CD4+ AIM+ response lower than one-half the median (<3.7% CD4+ AIM+ of total CD4+ T cells) were eliminated from the analysis. T cell data was analyzed using FlowJo 10.7.1. All data was background subtracted. Statistical analysis was carried out with GraphPad Prism 5 (GraphPad Software). Differences between groups were tested by the 2-tailed Fisher's exact test or Mann-Whitney test, with a p<.05 being considered significant. Correlation graphs were analyzed using Spearman's Correlation test. All volunteers in this study provided informed consent for the IRB approved study (University of Maryland, Baltimore). Correlation between AIM + (surface CD40L + OX40 + ) and CD40L + IFNγ + CD4 + T cells in (A) Antibody negative and (B, C) SARS2 exposed individuals. Statistics were calculated using Spearman's correlation. Supplemental Figure 9 . Correlation between pre-existing spike-specific CD4 + T cells and neutralization. Correlation between pre-existing spike-specific AIM + (surface CD40L + OX40 + ) CD4 + T cells (panels A,C,E) or AIM + (surface CD40L + OX40 + ) cT FH cells (panels B,D,F) and ID99 neutralization prior to vaccination, 14 days after 1 st vaccination, and 14 days after 2 nd vaccination. Asymptomatic and Symptomatic groups shown in blue and black circles, respectively. Statistics were calculated using Spearman's correlation.

A.

C.

D.

Pre-vaccination AIM + (CD40L + OX40 + ) CD4 + T cells (%) Log ID99 neutralization titer 

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ADH supported by CDC grant U01CK000556-02-01. This work was supported in part by Merit Award #