key: cord-0834274-85d4rvf1
authors: Pallikkuth, Suresh; Williams, Erin; Pahwa, Rajendra; Hoffer, Michael; Pahwa, Savita
title: Association of Flu specific and SARS-CoV-2 specific CD4 T cell responses in SARS-CoV-2 infected asymptomatic heath care workers
date: 2021-08-30
journal: Vaccine
DOI: 10.1016/j.vaccine.2021.08.092
sha: e2526ce85107d2929506d8dd990330771f04a938
doc_id: 834274
cord_uid: 85d4rvf1

Influenza vaccination is widely advocated to avoid infection with influenza virus, a serious respiratory pathogen, and this was greatly emphasized during the raging COVID-19 epidemic. We conducted a study for baseline Flu specific immunity in a group of health care workers with documented past SARs-CoV-2 infection (designated COVID+) with mild or no symptoms and compared them with a control group that had not been infected with SARS CoV-2 (COVID-). Concurrently, we examined flu and SARS-CoV-2 specific T cell responses using the AIM (activation induced molecules) assay by flow cytometry. All COVID+ and 40% COVID- participants exhibited AIM responses to SARS-CoV-2 peptides, but only COVID+ were positive for SARs-CoV-2 antibody. Influenza HIN1 antigen specific CD4 T cells were found in 92% COVID+ and 76% COVID- participants and exhibited a strong direct correlation with SARS-CoV-2 specific CD4 T cells. This observation suggests that influenza specific T cell immunity may impact immune responses to SARS-CoV-2.

According to CDC estimates, the 2018-2019 influenza season in the US was associated with over 35 million illnesses, over 16 million medical visits, close to 500,000 hospitalizations, and 34,200 deaths. Global estimates are that infection with Influenza virus leads to approximately 650,000

deaths each year. This topic achieves greater prominence during the COVID19 pandemic as it was unclear how the two respiratory infections with some overlapping symptomology will influence each other. Infections with human circulating "common cold" coronaviruses (HCoVs), such as HCoV-229E, -OC43, -NL63, and -HKU1 cause 15%-30% of milder common colds in adults [1] . Unlike SARS-CoV and influenza viruses that spread from the upper airway to cause a severe lower respiratory tract infection, HCoVs replicate principally in the upper respiratory tract (URT) epithelial cells, to cause local respiratory symptoms. It is currently not known whether coinfection with influenza or other HCoVs impact COVID-19 disease outcomes.

Emerging data suggests that SARS-CoV-2 specific CD4 and CD8 T cells are identifiable in a subset of people without evidence of active infection with or antibody directed against SARS-CoV-2 [2] [3] [4] . This immune response against SARS-CoV-2 has been attributed to preexisting immunologic memory against HCOVs that is cross-reactive for SARS-CoV-2 because of partial sequence homology of HCOVs with SARS-CoV-2 [4] . We questioned whether immunity to influenza virus, an unrelated respiratory pathogen, could influence immunity to SARS-CoV-2 through a mechanism such as "trained immunity". Reports that BCG and MMR vaccination could confer an immunologic benefit to some persons with SARS-CoV-2 supported this concept [5] [6] [7] [8] [9] [10] [11] [12] . In this study, we investigated Flu H1N1 antigen specific T cell responses in conjunction with SARS-CoV-2 specific T cell responses. The study was conducted in study participants during June-August 2020 with and without recent documented asymptomatic COVID-19 but prior to the onset of the 2020-2021 flu vaccination season. Two key observations were made.

First, in agreement with published reports [2] [3] [4] 13] , a significant proportion of COVID Ab negative persons without history or evidence of SARS Cov-2 infection exhibited demonstrable SARS-CoV-2 specific CD4 T cell immune responses. Second, a novel finding was that SARS-CoV-2 specific CD4 T cells were strongly correlated with H1N1 antigen specific CD4 T cells.

Study groups: We identified participants with prior SARS-CoV-2 infection confirmed by SARS-CoV-2 DNA+ (n=21) during June 2020 -August 2020. All COVID+ participants were health care workers employed at the University of Miami and are a subset of participants from a larger cohort of COVID immunity study. All had mild/moderate symptoms without hospitalization. The median age of COVID+ participants was 34.5 yrs (range: 27-61) with 52% (11/21) females. A group of SARS-CoV-2 seronegative community participants (n=33) was 35 yrs (range: 24 -77) with 51.5% (17/33) females were included as COVID-group. A summary of the demographic characteristics and influenza vaccination history of the study groups is shown in Table 1 . Antibody response to flu measured as hemagglutination inhibition (HAI) titers to H1N1 flu antigen at the study entry did not differ between the groups but a trend of higher response (P =0.054) was noted in the COVID+ group (not shown). Peripheral venous blood was collected after obtaining written informed consent and peripheral blood mononuclear cells (PBMC) were separated and cryopreserved in liquid N 2. This study was approved by the Institutional Review Boards of the University of Miami.

Antigens used for the stimulation experiments: Megapools specific to SARS-CoV-2spike (CD4S) and non-spike (CD4R), CD8 A and CD8 B were provided as a gift by Dr. Sette, UCSD, La Jolla, CA. Peptide sequences and the effectiveness of these peptides to induce SARS-CoV-2 specific responses have been reported in previous by Sette lab [2, 4] and recently by a collaborative study between Sette group and our lab [14] . Briefly, SARS-CoV-2 virus-specific CD4 and CD8 peptides were synthesized as crude material, resuspended in DMSO, pooled and sequentially lyophilized. SARS-CoV-2 epitopes were predicted using the protein sequences derived from the SARS-CoV-2 reference (GenBank: MN908947) and IEDB analysis-resource.

MPs were generated based on the predicted SARS-CoV-2 epitopes. CD4_R MP corresponds to 221 predicted HLA class II CD4+ T cell epitopes covering all proteins in the viral genome, apart from the spike (S) antigen ( n=221 peptides). For the MP_S, a separate MP containing 253 peptides covering the entire antigen with 15-mer peptides overlapping by 10-residues was used.

CD8 SARS-CoV-2 epitope prediction was performed for the top 12 more frequent HLA alleles and the resulted 628 predicted CD8 epitopes were split in two CD8 MPs containing 314 peptides each (CD8-A and CD8-B) [2, 4] . The H1N1 antigen used in this study was a purified, formalin inactivated, whole virus preparation obtained from the Center for Biologics Evaluation and Research (CBER), FDA that has been previously used for investigation of flu specific T cell responses [15, 16] . We and others have reported the use of inactivated, whole virus preparation for flu specific assays to capture the breadth of the antigen specific CD4 and CD8 T cell responses that have included intracellular cytokine secretion (ICS) as a functional readout [15] [16] [17] [18] Antigen induced activation molecule (AIM) assay: Thawed PBMC were cultured for 24 hours in the presence of 4 different SARS-CoV-2 specific megapools 2 each for CD4 and CD8 T cells When two stimuli were combined for calculating the total SARS-CoV-2 specific responses, the percentage of AIM+ cells after SARS-CoV-2 stimulation were added together and the value was then subtracted by twice the value of the percentage of AIM+ cells derived from DMSO stimulation. A p value <0.05 was significant.

Antigen specific CD4 T cells were identified as CD137+OX40+ CD4 T cells (Fig 1A) or as CD69+OX40+ CD4 T cells (Fig 1B) . SARS-CoV-2 spike (S), SARS-CoV-2 non-spike (R) and total response calculated by adding the frequencies of spike and non-spike specific CD4 T cells together were present in 100% post-COVID volunteers (Fig 1A, 1B) . In line with recent reports [2, 4, 14] nearly 40% COVID-participants had detectable SARS-CoV-2 specific CD4 T cells above the background levels with a stimulation index ≥ 3 (Fig 1C, 1D) .

H1N1 specific AIM+ CD4 T cells above background levels were detected in 92% COVID+ and 55% of COVID-participants (Fig 1A, 1B) while SI above 3 for H1N1 response was found in 92% COVID+ and 76% of COVID-participants (Fig 1C, 1D) . A strong direct correlation was observed between H1N1 specific OX40+CD137+ CD4 T cells with total SARS-CoV-2 specific CD4 T cells (Fig 2A) , individual CD4 R (Fig 2B) and CD4 S (Fig 2C) peptide specific CD4 T cells in both COVID+ and COVID-groups. Similar direct correlations were found between H1N1 specific CD69+OX40+ CD4 T cells and SARS-CoV-2 specific CD69+OX40+ CD4 T cells in both the groups (Fig 2D-E) . In the COVID+ cohort, additional activation induced marker combinations OX40+CD25+ and CD40L+CD69+ were also detected on CD4 T cells following antigen stimulation, and they correlated each other (Fig 3) .

CD8 T cell responses: We next analyzed the relationship between H1N1 specific CD8 T cells and SARS-CoV-2 specific CD8 T cells in this cohort based on the co-expression of CD69 and CD137 [14] . As expected, SARS-CoV-2 specific CD8 T cell responses were significantly higher in COVID+ group compared to the COVID-group. SARS-CoV-2 specific CD8 T cells above the background levels were less frequent in the seronegative participants with 9% response and stronger in the COVID+ participants with 95% response. Stimulation index above 3 for SARS-CoV-2 specific CD8 T cells was found in 9% seronegative and 66% in COVID+ participants.

Frequencies of H1N1 specific CD8 T cell response were not significantly different between COVID+ and COVID-participants although a trend of higher frequencies was noted in COVID+ participants (Fig 4A) . H1N1 specific CD8 T cell response above background levels were seen in 42% COVID-and 95% COVID+ participants while SI of CD8 T cell response above 3 was noticed in 79% of COVID-and 95% COVID+ participants. Correlation analysis did not show any association between H1N1 specific CD8 T cell responses with SARS-CoV-2 specific CD8 T cell responses in either COVID+ or COVID-participants (Fig 4B) .

Taken together, our data support a strong association of antigen specific CD4 T cell responses for Flu and SARS-CoV-2 that was evident in both COVID+ and COVID-participants with CD4

T cell responses to SARS-CoV-2 in 40 % of the COVID-groups. Although CD8 T cell responses were higher in COVID+ participants, the CD8 T cell response did not show any association with Flu response in either group.

The goal of this study was to investigate if immune responses to H1N1 influenza virus antigen were also associated with SAR-CoV-2 cellular immune responses that were detected in people without serologic evidence of prior CoV-2 infection as compared to those with documented prior CoV-2 infection (PCR confirmed and IgG Ab positive). We have recently reported the finding that SARS CoV-2 specific CD4 T cell immunity is detectable in >50% of a group of high-risk healthcare workers who did not have prior SARS CoV-2 infection and were seronegative while 96 % of people with past documented COVID-19 who were also seropositive had strong CD4 T cell immunity [14] . As this study was conducted during June-August 2020, prior to the 2020-2021 influenza vaccination period we investigated the same participants for pre-existing influenza memory responses and serology. A strong relationship was observed between CD4 T cell activation induced markers in response to influenza virus H1N1 antigen and SARS-CoV-2 antigens. We contend that pre-existing immunity to Influenza may affect the immunity to SARS-

There is growing consensus that pre-existing immunity against other respiratory pathogens may enhance the COVID-19 specific immunity through a mechanism entailing cross reactive antigens. This concept is supported by the fact that more than 90% of the human population is seropositive for at least three of the HCoVs [13] and the reported T cell reactivity thus far was highest against a pool of SARS-CoV-2 spike peptides that had significant homology of homology to HCoVs [13, 19] . Findings from this study point to a relationship of immunity against influenza with immunity against SARS-CoV-2. A strong relationship of Flu H1N1 specific CD4 T cell responses with the SARS-CoV-2 specific CD4 T cell response was observed that was absent for the CD8 T cell compartment. Three different combinations of AIM markers on CD4 T cells signifying memory cell activation showed similar correlations solidifying the consistency of this observation. The underlying mechanism for the observed association of cellular CD4 T cell responses against flu and SARS-CoV-2 is not known but several possibilities exist. There is data supporting cross reactivity of immunity between Flu and coronavirus [20] due to the similarity in their viral envelope glycoprotein hemagglutinin-esterases (HE) that mediate virion attachment, receptor destruction, and membrane fusion [21] . The most plausible explanation for the T cell immunity to Flu is that of repeated past vaccinations, which is of particular relevance in this participant group comprised of health care workers. 95% of our COVID+ participants were vaccinated with seasonal flu vaccine in the 2019-2020 influenza season, prior to becoming in infected with SARS-CoV-2, implying pre-existing H1N1 specific memory T cells in this cohort. Further studies are needed to understand the direct effect of the immune responses associated with SARS-CoV-2 infection on the flu specific T cell responses in our study participants. A previous study reported an increased non-specific reactivity of PBMC of COVID-19 recovered patients to unrelated antigenic stimulation [22] . However, higher nonspecific activation of T cells was not evident in our participants as we did not find significant differences in the activation status of the CD4 and CD8 T cells at baseline or after 24 hours of H1N1 stimulation measured as frequencies HLA-DR+CD38+ cells (not shown). We also did not find any significant alterations in the frequencies of CD4 or CD8 T cells or their maturations subsets between the study groups (not shown). Moreover, the association of SARS-CoV2 specific response was noted only for flu H1N1 antigen but not for CMV peptide-or generalized (anti-CD3+CD28) stimulation (not shown). Although less likely, the possibility exists that the

Influenza.

We favor the contention that flu specific immunity may also induce bystander immunity analogous to "trained immunity" that can non-specifically augment T cell responses against SARS-CoV-2. The concept of trained immunity is that the long-term functional reprogramming of innate immune cells evoked by exogenous or endogenous stimulation leads to an altered response towards a second heterologous challenge [23] . Epidemiological data supports the concept that live vaccines such as the BCG, measles, smallpox and oral polio have beneficial, non-specific protective effects against infections other than the target diseases [23] . BCG vaccination led to protection against microorganisms in models of controlled human infection, such as yellow fever or malaria, and this was associated with an augmented proinflammatory activity of monocytes [24, 25] Our study is limited by number of study participants, reliance only on AIM markers for immune assessment and use of whole inactivated H1N1 virus as antigen for CD8 T cells that may not capture all specific CD8 epitopes.

We speculate a possible benefit of the seasonal flu vaccination as being favorable for SARS-CoV2 specific T cell immunity. Regardless of the basis for the augmentation of T cell responses directed against SARs-CoV-2, flu specific immunity may influence the immune response to COVID-19 in this study population. responses. Data were expressed as Mean ± Standard Deviation (SD) and Kruskal-Wallis test with Dunn's multiple comparisons was used for comparing between group. Line with stars indicates difference between 231 time points within a group and between groups and the level significance as *p<0.05; **p<0.01; ***p<0.001 A p value of <0.05 was considered as significant. Kruskal-Wallis test with Dunn's multiple comparisons was used for comparing between group.

For correlation analyses, non-parametric spearman correlation was used. A p value of <0.05 was considered as significant.

Characteristics of SARS-CoV-2 and COVID-19

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Cross-reactive memory T cells and herd immunity to SARS-CoV-2

Selective and crossreactive SARS-CoV-2 T cell epitopes in unexposed humans

Vaccine repurposing approach for preventing COVID 19: can MMR vaccines reduce morbidity and mortality?

MMR Vaccination: A Potential Strategy to Reduce Severity and Mortality of COVID-19 Illness

Analysis of Measles-Mumps-Rubella (MMR) Titers of Recovered COVID-19 Patients Could an Unrelated Live Attenuated Vaccine Serve as a Preventive Measure To Dampen Septic Inflammation Associated with COVID-19 Infection? mBio

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Pre-existing immunity to SARS-CoV-2: the knowns and unknowns

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HIV infection Worsens Age-Associated Defects in Antibody Responses to Influenza Vaccine

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Defining trained immunity and its role in health and disease

BCG Vaccination Protects against Experimental Viral Infection in Humans through the Induction of Cytokines Associated with Trained Immunity

Outcomes of controlled human malaria infection after BCG vaccination

Median Spike IgG Titer, Median (range)

Flu Vaccinated, n (%)

We thank Dr. Alessandro Sette and Dr. Daniela Weiskopf from La Jolla Institute for Immunology for providing SARS-CoV-2 specific peptides and creative input for the experimental design. We also acknowledge Dr. Shane Crotty for the thoughtful insight about data analysis. We thank the Clinical Research Center at the University of Miami for the blood draw. We also thank Erin Williams for the study co-ordination and recruitment, Margaret Roach, Elizabeth Varghese, Maria Pallin and Dan Kvistad from the Pahwa lab for their assistance with sample processing and laboratory experiments and to participants of this study.Funding: This work was supported by National Institutes of Health Grant: R01AI108472 (to S.P.).

The authors declare no competing interests.