Method for in vitro evaluation of the immune response after vaccination with a mRNA vaccine

The present invention concerns a method for in vitro evaluation of the immune response after a mRNA vaccination. In particular, the present invention concerns a method for in vitro evaluation of the immune response after a mRNA vaccination by detecting specific biomarkers.

It is well known that mRNA vaccines have been proved to induce both humoral and cellular immune responses in prophylactic and in treatment settings. In some responders, clinical responses are associated with strong and durable CD4+ and CD8+ T cell responses against the vaccine antigens(7-4).

In the last two years, we have been facing one of the worst pandemics in our history since the last post-war period caused by SARS-CoV-2, a new coronavirus(5), whom infection has caused remarkable morbidity and a dramatic impact in terms of human lives. Although some symptoms share this pathogen with the previous coronaviruses responsible for SARS (severe acute respiratory syndrome) and MERS (middle East respiratory syndrome)(6), this new virus is characterized by a remarkable transmissibility, high viremia even in asymptomatic subjects, and a lower mortality rate (6-9).

So far, the World Health Organization (WHO) has registered nearly 506 million cases of infection, with more than 6.2 million deaths. The circulation of the virus could be kept under control by herd-immunity, achieved through both natural SARS-CoV-2 infection or vaccination. While the first mRNA BNT162b1 vaccine encoded for the spike RBD domain (222 aa), the current BNT162b2 mRNA vaccine codifies for the full-length SARS-CoV-2 Spike protein (1273 aa) (1, 10) derived from the ancestral strain identified in Wuhan (China) (11). RBD, which contains a core and a receptor-binding motif (RBM) which mediates contacts with the ACE2 receptor (12), is highly immunogenic and conserved among human Sarbecoronavirus, SARS-CoV and SARS-like viruses and emerged variants of concern (VOC) (13).

The immunogenicity, long term efficacy and widespread protective immunity represent key hallmarks of successful vaccination. Understanding immune responses to SARS-CoV-2 mRNA vaccines is of great interest, principally because of the poor knowledge about the mechanisms of immune protection, quality and durability of the induced adaptive immune responses against the ancestral virus and the emerging variants of concern (VOC).

The administration of the new mRNA vaccines against SARS-CoV-2 is having an important impact in the control of the virus spreading, however the low incidence of SARS-CoV-2 infections among vaccinated subjects at the early stages following the mRNA vaccine administration, is loss some months after the first two dose vaccination (14, 15).

Following SARS-CoV-2 mRNA vaccination, memory B, CD4 ⁺ and CD8 ⁺ T cells play a critical role in the protection against infection, and represent key determinants in the re-challenge and boost with the vaccine (11, 16), as well as protection after recovery from SARS-CoV-2 infection (17). Results obtained with both mRNA Pfizer/BioNTech and Moderna vaccination reached 91 % and 93% protective immunity over 7 months after first immunization (10, 14, 18), and up to 95% efficacy in preventing symptomatic COVID-19 disease (2, 19). Based on these considerations, understanding of the mechanisms of protective memory responses would allow to develop new protocols of vaccination, to monitor vaccinated subjects and would contribute to the development of new vaccines (20).

Humoral immunity can persist for more than 8 months after the first two mRNA vaccine injections (21, 22). However, a decline of the circulating antibodies which are produced by short-term plasmablasts, have been associated with higher risk of infections, although with less chances to develop severe COVID-19, hospitalization and death (15, 23), likely because of the maintenance of specific memory B and T cells promptly responding and differentiating on demand (i.e. , after antigen re-encounter) (24, 25). Long-lived memory B cells can undergo antigenbinding receptor hypermutations that promote generation of higher affinity antibodies (25), including those specific for RBD, a key target of neutralizing antibodies, generating durable immunological memory. Based on these considerations, a third dose of vaccine has been recommended (WHO).

In the case of subjects infected with SARS-CoV-2, natural immunity has been reported 93% to 100% protective against symptomatic disease for at least 7-8 months (26-28). As a consequence, the individuals resolving prior SARS-CoV-2 infection, have been also vaccinated between six months and one year after infection to achieve long-term protection (hybrid immunity) (29) (30, 31). Recent observations indicate that hybrid immunity elicits levels of cross-variant neutralizing antibodies significantly higher and more resistant than those obtained by immunization of naive donors (29) (30, 31). This strong immunity may be related with the observation that memory B cells display higher clonal turnover, greater somatic hypermutation, more resistance to RBD mutations and increased potency, related to the continuous evolution of the humoral response in SARS-CoV-2 recovered individuals as compared to vaccinated naive ones (32, 33). Nevertheless, while several studies have clearly reported the role of virus-specific B and T cells in contributing to protective immunity or immunopathology in SARS-CoV-2 vaccinated, recovered, or infected individuals (7, 34, 35) (31, 36), it is still unclear if hybrid immunity generates more robust repertoire of long-lived memory B and T cell responses conferring super-immunity, as compared to immunity developed in SARS-CoV-2 naive subjects following vaccination alone.

Therefore, while recent findings indicate durable virus-specific B cell responses following vaccination ^{18 19} , it is still unknown how to evaluate the impact of this new mRNA vaccine on the induction of poly-specific CD8+ T cell subsets against the new variants and on the generation of long-lasting spike-specific antibodies, and as a consequence the host protection against COVID-19.

In particular, the emergence and circulation of the new VOC, including B.1.1.7 (Alpha), B.1.351 (Beta), P1 (Gamma) and B.1.617.2 (Delta), and the most recent B.1.1.529 (Omicron), have shaded light on the poor knowledge on the longterm efficacy of protection of the mRNA vaccines against the new viruses, and on the quality and long-term persistence of cross-reactive memory CD4 ⁺ and CD8 ⁺ T cells (11), (16, 37-39). Moreover, while long-term memory B cell and CD4 ⁺ T cell responses to the mRNA vaccine against SARS-CoV-2 start to be elucidated (21, 22, 40), the diversification of memory CD8 ⁺ T cell responses are not completely understood.

It is known that CD8 ⁺ T cells comprise several memory subsets, including CM, EM1 , EM2, EM3 and terminally differentiated effector memory CD45RA (EMRA) (41-43). The CD8 ⁺ TCM, and TEM subsets are also characterized by distinct transcriptional and epigenetic programs (44). The expression level of CCR7 and CD27 characterizes CD8 ⁺ T cell differentiation, the stepwise loss of these surface markers guides CD8 ⁺ T cell commitment (41). While EM3 cells mostly resemble differentiated EMRA cells, EM1 cells are closely related to CM cells. EM1 subset has been suggested to exert memory functions in peripheral tissues, while CM cells reside in lymphoid organs (41). So far, fundamental issues about the mechanisms of CD4 ⁺ and CD8 ⁺ T cell differentiation after mRNA vaccination, the dynamics of T cell subsets, their turnover rate in vivo, and efficacy of protection against SARS-CoV-2, remain open questions.

Currently, the immune response induced in individuals who received a mRNA vaccination, such as a mRNA vaccination against SARS-CoV-2, can be evaluated by measuring the antibody titer and neutralizing activity of IgG against the antigen targeted by the vaccine, for example the antibody titer of anti-Spike IgG. However, this type of measurement does not allow to evaluate the protective immunity against the infection and against the possible development of a related disease, such as COVID-19, or, in case of anti-tumor vaccines, against the tumor. Moreover, the protective immunity mediated by CD8+ T cells after the administration of mRNA vaccine is still poorly understood, and not yet correlated with protective immunity.

In the light of the above, it is therefore apparent the need to provide new methods to evaluate the quality of immune response after a vaccination with mRNA, which overcome the disadvantages of the known methods.

According to the present invention a new method able to evaluate humoral and memory CD8+ T cell responses induced by mRNA-based vaccines and to distinguish high responders and low responders to a mRNA vaccination, such as SARS-CoV-2 vaccination, is provided. The method according to the present invention is advantageously able to identify those subjects more susceptible to the infections or diseases, such as tumor, against which the vaccination is directed, therefore contributing to design new vaccination and booster protocols.

In particular, as shown in the experimental data reported below, the durability of the spike-specific antibodies induced by mRNA vaccine, in correlation with circulating memory B cells and CD8+ T cells, in both SARS-CoV-2-vaccinated naive (SARS-CoV-2-N) and SARS-CoV-2-vaccinated ex COVID-19 recovered (SARS- CoV-2-EC) individuals, has been analyzed. The investigations have been also extended to the analysis of CD8 ⁺ T responses against the most widespread variants of concern (VOC). In particular, the binding antibody and CD8+ T cell memory programs against wild type SARS-CoV-2 and B.1.1.7 (Alpha), B.1.351 (Beta), P1 (Gamma) and B.1.617.2 (Delta) and B.1.1.529 (Omicron), VOC were longitudinally analysed over 6 months post-immunization with a mRNA-based vaccine.

More in detail, according to the experimental data reported below, SARS- CoV-2-N individuals have been distinguished in high and low responders (HRs and LRs) to mRNA vaccination, according to the antibody titers and distinct memory CD8 ⁺ T cell subsets reactive against spike.

According to the present invention, it has been found that the magnitude and the mechanisms of CD8+ T cell mediated-immunity can change during the evolution of the responses from the baseline, peak, contraction, and memory phase.

In addition, as shown in the experimental data reported below, it has been observed that the responses of SARS-CoV-2-N HRs were principally characterized by the activation of CM and EM1 CD8 ⁺ T cells and resemble to those from previously infected individuals (hybrid immunity), principally distinguished by CM CD8+ T cell responses. Conversely, SARS-CoV-2-N LR responses were mainly associated with EM3 activation. Opposite to the low responders, the high responders were characterized by increased frequency of central memory (CM) and effector memory

1 (EM1 ) CD8+ T cells, sustained T cell effector function and durable SARS-CoV-2 spike-specific responses, with diverse reactivity against the spike receptor binding domain (RBD) derived from the most widespread VOC. The vaccination of previously SARS-CoV-2 infected individuals boosted antibody levels and promoted durable specific CM CD8 ⁺ T cells.

Importantly, according to the present invention it has been shown that the distinct adaptive immune responses induced by RNA vaccine correlated with long- lasting protection against COVID-19 in SARS-CoV-2-N HRs and SARS-CoV-2-EC subjects (ECs), but not in SARS-CoV-2-N LRs, also after the complete cycle of vaccination (three mRNA doses). In fact, low responders were less protected against COVID-19 after the second and the third dose of mRNA vaccine.

Therefore, the results described above demonstrate that mRNA vaccine induces long-term specific memory CD8+ T cells with distinct poly-reactive properties against SARS-CoV2 and VOC, thus affecting long-term protection against COVID-19 in high, low responders and vaccinated individuals with preexisting immunity.

As stated above, the results obtained according to the present invention unravel three main categories of responders to mRNA vaccination, on the basis of anti-spike IgG antibodies and poly-reactive memory CD8 ⁺ T cells, with important implications for the identification of subjects more or less susceptible to SARS-CoV-

2 infection and development of COVID-19. The distinct phenotype and frequency of the memory subsets observed in the SARS-CoV-2-N LRs correlate with reduced efficacy of protection against COVID-19 development after SARS-CoV2 infection.

On the basis of the activation and memory markers of CD8+ T cells, possibly in combination with the anti-spike antibody titer, a new method to distinguish high and low responders to mRNA vaccination is now provided according to the present invention.

The method according to the invention can be advantageously used to evaluate the responses induced by a mRNA vaccine of the most fragile people, including elderly, subjects affected by chronic disease, including patients with cancer and immunodeficiencies, and to define the time schedule for the third and following boost-dose administration.

In particular, the method of the present invention can allow to determine the long-term efficacy of the vaccine, to monitor the specific long term memory responses, to design new vaccination and targeted boost follow-up protocols for the different responder categories, especially for the most fragile people, the most susceptible to SARS-CoV-2 infection and COVID-19.

The results obtained according to the present invention on biological samples of subjects vaccinated with anti-SARS-CoV-2 mRNA vaccines make it plausible for the person skilled in the art that similar comparable results can be achieved on biological samples of subjects vaccinated with other mRNA vaccines, since the mRNA vaccine are designed to target antigen presenting cells, in these cells the mechanisms of antigen synthesis and processing for these vaccines are always the same. In fact, vaccination with mRNA against an antigen is based on the use of a synthetic mRNA molecule produced in the laboratory and coding for an antigen of interest. Since the vaccination is with a synthetic molecule, the type and quality of the response to mRNA vaccines will follow the same biological processes, such as antigen presentation and T lymphocyte activation, which will lead to the development of the memory immune response.

It is therefore specific object of the present invention a method for in vitro evaluation of the immune response to an antigen (i.e. one or more antigens) or a variant thereof or to an immunogenic portion of said antigen or variant thereof after the administration of a mRNA vaccine to a subject, wherein the mRNA vaccine encodes said antigen, said method comprising: a) detecting, in a biological sample of said subject, collected after the administration of said mRNA vaccine, CD8+ T cells, i.e. cells expressing CD8 marker, for example by means of a labelled anti-CD8 antibody, such as a fluorescent anti-CD8 antibody, or by means of primers and/or probes; b) measuring or obtaining a measurement of the basal expression of CD25 marker in the CD8+ T cells, for example by means of a labelled anti-CD25 antibody, such as a fluorescent anti-CD25 antibody, or by means of primers and/or probes; c) putting the CD8+ T cells in contact with the antigen or a variant thereof or the immunogenic portion of said antigen or variant thereof and measuring or obtaining a measurement of the expression of CD25 marker in the CD8+ T cells, for example by means of a labelled anti-CD25 antibody, such as a fluorescent anti- CD25 antibody, or by means of primers and/or probes; wherein when the expression of CD25 marker measured in step c) is higher than the basal expression of CD25 marker measured in step b), the subject presents an immune response to the antigen or variant thereof or to the immunogenic portion of said antigen or variant thereof, whereas when the expression of CD25 marker measured in step c) is equal or lower than the basal expression of CD25 marker measured in step b), the subject does not present any immune response to the antigen or variant thereof or to the immunogenic portion of said antigen or variant thereof.

According to the method of present invention, said step c) can comprise an incubation time after putting the CD8+ T cells in contact with the antigen, or a variant thereof or the immunogenic portion of said antigen or variant thereof, and before measuring the expression of CD25 marker, wherein said incubation time can range from 12 to 72 hours, preferably from 16 to 48 hours, more preferably 24 hours.

The mRNA vaccine according to the present invention can be a preventive mRNA vaccine, such as an anti-viral or anti-bacterial mRNA preventive vaccine, or a therapeutic mRNA vaccine, such as an anti-tumor mRNA vaccine.

According to the present invention, said subject can be a human subject or an animal subject, such as a mouse. The method according to the present invention can be used for validating a mRNA vaccine, for example by applying the method of the invention in animal subjects, such as mice, in order to evaluate the immune response induced by a new mRNA vaccine.

The method according to the present invention is able to evaluate the immune response induced by the same antigen (or antigens) encoded by the mRNA vaccine or, in particular when the vaccine is against a virus or a bacterium, to variants of the antigen encoded by the mRNA vaccine or to immunogenic portions of said antigen or variant. For variant is intended a mutated antigen characterized by one or more aminoacidic substitutions.

For example, if the mRNA vaccine encodes for the Spike protein of SARS- CoV-2 virus, the method of the invention can evaluate the immune response of the vaccinated subject to the same Spike protein of SARS-CoV-2, or it can evaluate the immune response of the vaccinated subject to a variant of Spike protein of a SARS- CoV-2 variant, such as Alpha, Beta, Gamma, Delta or Omicron variants. In addition, the method of the invention can evaluate the immune response of the vaccinated subject to an immunogenic portion of Spike protein of SARS-CoV-2 or variants thereof, such as RBD immunogenic portion.

In order to evaluate the quality of the immune response, in particular the protective immune response, in subject who presents an immune response to the antigen or variant thereof or to the immunogenic portion of said antigen or variant thereof, the method according to the present invention, when the subject presents an immune response to the antigen or variant thereof or to the immunogenic portion of said antigen or variant thereof, can further comprise: d) detecting CD45RA- CD8+ CD25+ T cells among CD8+ CD25+ T cells detected above, for example by means of a labelled anti-CD45RA antibody, such as a fluorescent anti-CD45RA antibody, or by means of primers and/or probes; therefore, CD8+ CD25+ T cells which do not express CD45RA marker (CD45RA-) are detected, which comprise memory T cells; e) detecting the expression of CD27 marker in the detected CD45RA- CD8+ CD25+ T cells, for example by means of a labelled anti-CD27 antibody, such as a fluorescent anti-CD27 antibody, or by means of primers and/or probes; wherein said step e) of detecting the expression of CD27 marker can be carried out before, after or together with said step d), and wherein, when CD27 marker is expressed in said CD45RA- CD8+ CD25+ T cells (i.e. when the T cells are CD27+ CD45RA- CD8+ CD25+), said subject presents an high response to the antigen or variant thereof or to the immunogenic portion of said antigen or variant thereof (high responder), whereas when CD27 marker is not expressed in said CD45RA- CD8+ CD25+ T cells (i.e. when the T cells are CD27- CD45RA- CD8+ CD25+),said subject presents a low response to the antigen or variant thereof or to the immunogenic portion of said antigen or variant thereof (low responder).

Protective immune response is intended as the immune response by which infection is prevented or, if infection occurs, by which severe symptoms are prevented and only mild symptoms occur.

According to the present invention, a subject who presents a high response to the antigen or variant thereof or to the immunogenic portion of said antigen or variant thereof (high responder) is intended as a subject who has a lower risk of developing the disease against which the subject has been vaccinated through said mRNA vaccine. For example, if the mRNA vaccine is against a virus, such as SARS- CoV-2 virus, a high responder subject has a lower risk of developing a disease caused by the infection of said virus, such as COVID-19 disease. If the mRNA vaccine is against a bacterium, a high responder subject has a lower risk of developing a disease caused by the infection of said bacterium . If the mRNA vaccine is a therapeutic vaccine against a tumor, i.e a vaccine administrated in a subject having a tumor, a high responder subject has a lower risk of disease progression and a higher probability of recovering from said tumour. If the mRNA vaccine is a preventive vaccine against a tumor, such as a prophylactic vaccine administrated to a subject having a genetic predisposition to develop a tumour, for example breast tumour, a high responder subject has a lower risk of developing the tumour.

According to the present invention, a subject who presents a low response to the antigen or variant thereof or to the immunogenic portion of said antigen or variant thereof (low responder) is intended as a subject who has a higher risk of developing the disease against which the subject has been vaccinated through said mRNA vaccine. For example, if the mRNA vaccine is against a virus, such as SARS-CoV- 2 virus, a low responder subject has a higher risk of developing a disease with mild/severe symptoms caused by the infection of said virus, such as COVID-19 disease. If the mRNA vaccine is against a bacterium, a low responder subject has a higher risk of developing a disease caused by the infection of said bacterium. If the miRNA vaccine is a therapeutic vaccine against a tumor, i.e. a vaccine administrated in a subject having a tumor, a low responder subject has a higher risk of disease progression and a lower probability of recovering from said tumor. If the mRNA vaccine is a preventive vaccine against a tumor, such as a prophylactic vaccine administrated to a subject having a genetic predisposition to develop a tumour, for example breast tumour, a low responder subject has a higher risk of developing the tumour.

The method according to the present invention can be advantageously be used for evaluating the effectiveness and the duration of the effectiveness of mRNA therapeutic vaccine, such as an anti-tumor mRNA vaccine, and modifying the therapy in case of non-responder or low responder subjects.

In addition, the method according to the present invention can be advantageously used for evaluating the effectiveness of a mRNA preventive vaccine, such as an anti-viral or anti-bacterial mRNA vaccine, and modifying the number of booster doses or modifying the type of preventive vaccine in case of non- responder or low responder subjects.

In order to further evaluate the quality of immune response to the vaccine in high responder subjects, the method according to the invention, when said subject presents a high response to the antigen or variant thereof or to the immunogenic portion of said antigen or variant thereof, can further comprise a step f) of detecting the expression of CCR7 marker in CD27+ CD45RA- CD8+ CD25+ T cells, for example by means of an anti-CCR7 antibody, such as a fluorescent anti-CCR7 antibody, or by means of primers and/or probes; wherein when CCR7 marker is expressed in said CD27+ CD45RA- CD8+ CD25+ T cells (i.e. when the T cells are CCR7+ CD27+ CD45RA- CD8+ CD25+), said subject has central memory (CM) T cells and has a higher immune response to the antigen or variant thereof or to the immunogenic portion of said antigen or variant thereof in comparison to when CD27 marker is not expressed in said CD45RA- CD8+ CD25+ T cells (i.e. when the T cells are CD27- CD45RA- CD8+ CD25+), i.e. when the subject has effector memory 1 (EM1 ) T cells.

According to the present invention, said antigen encoded by said mRNA vaccine can be selected from a virus antigen, such as a SARS-CoV-2 antigen, a bacterial antigen or a tumour antigen.

Therefore, according to the present invention, the mRNA vaccine can be a vaccine against a virus infection and related diseases, against a bacterial infection and related diseases or against a tumor.

According to the present invention, said biological sample can be a biological sample collected starting from three months after the administration of said mRNA vaccine. For example, when the mRNA vaccine is against SARS-CoV-2, the method according to the present invention can be applied on biological samples collected from three months after the administration of the first two doses of said mRNA vaccine to the third dose injection and/or several months after administration of the third dose (boost).

According to the present invention, said method can further comprise measuring or obtaining the measurement of the antibody titre of IgG against said antigen or variant thereof or said immunogenic portion of said antigen or variant thereof.

According to the present invention, said biological sample can be a sample of blood, plasma or a tissue sample, such as tissue samples containing immune cells isolated from secondary lymphoid organs or tissues.

It is a further object of the present invention a kit for in vitro evaluation of the immune response to an antigen (i.e. one or more antigens) or a variant thereof or an immunogenic portion of said antigen or variant thereof after the administration of a mRNA vaccine in a subject, wherein the mRNA vaccine encodes said antigen, said kit comprising means for detecting CD8 marker, such as an anti-CD8 antibody, for example a fluorescent anti-CD8 antibody, or specific primers and/or probes for detecting CD8 marker by means of PCR or qPCR, and CD25 marker, such as an anti-CD25 antibody, for example a fluorescent anti-CD25 antibody, or specific primers and/or probes for detecting CD25 marker by means of PCR or qPCR; said antigen or a variant thereof or said immunogenic portion of said antigen or variant thereof.

According to the present invention, said kit can further comprise means for detecting CD45RA marker, such as an anti-CD45RA antibody, for example a fluorescent anti-CD45RA antibody, or specific primers and/or probes for detecting CD45RA marker by means of PCR or qPCR, and CD27 marker, such as an anti-CD27 antibody, for example a fluorescent anti-CD27 antibody, or specific primers and/or probes for detecting CD27 marker by means of PCR or qPCR;

According to the present invention, said kit can further comprise means for detecting CCR7 marker, such as anti-CCR7 antibody, for example a fluorescent anti-CCR7 antibody, or specific primers and/or probes for detecting CCR7 marker by means of PCR or qPCR.

According to the present invention, said kit can further comprise a labelled anti-IgG antibody. According to an embodiment of the present invention, said kit does not comprise means, such as antibodies or specific primers and/or probes, for detecting other markers different from CD25, CD8, CD45RA, CD27 and/or CCR7.

The present invention now will be described by an illustrative, but not limitative way, according to preferred embodiments thereof, with particular reference to the examples and the enclosed drawings, wherein:

- Figure 1 shows the longitudinal analysis of humoral responses to BNT162b2 mRNA vaccine. (A-B) Time series plots of IgG values measured in (A left) SARS-CoV-2 N, seropositive (no history of infection but with low level of specific Ab against SARS-CoV-2 antigens, A middle), and (A, right) SARS-CoV-2 recovered subjects. Statistics were calculated using Wilcoxon signed-rank test, with Benjamini Hochberg correction for multiple testing. (B) Kernel density estimation for IgG distribution values at 3 months is shown. The curve is composed by 32768 points with a distance between adjacent points of 5.82e-03 AU/rnl. The total area under the density curve (A=1 ) is estimated through the Riemann Sum with a normalizing constant of 0.999801. (C) Box plots of IgG values in SARS-CoV-2 naive LRs and HRs at time 0, 3 weeks, 6 weeks, 3 months and 6 months. Statistics were calculated using Wilcoxon rank-sum test, with Benjamini Hochberg correction for multiple testing. Mode (straight line) and anti-mode (dotted line) are shown. Antimode was used to distinguish SARS-CoV-2 N subjects into high and low responders. (Statistics were calculated using Wilcoxon rank-sum test, with Benjamini Hochberg correction for multiple testing. *P < 0.05; **P < 0.01 ; ***P < 0.001 ; ****P < 0.0001.

- Figure 2 shows that BNT162b2 mRNA vaccine induces different levels of CD8 ⁺ T cell activation against S_RBD variants in HR, LR and EC subjects. Box plots showing the log of CD25 ⁺ CD8 ⁺ cell percentage on gated CD8 ⁺ T cells from LR, HR and EC subjects at time 0, 6 weeks, 3 and 6 months after 48h of incubation with Spike, S_RBD_WT (wild type) or S_RBD indicated variants, at 6 weeks and 6 months after 48h of incubation with S_RBD_B.1 .617.2, at time 0 and 6 months with S_RBD_B11529. Data are represented with background subtraction from paired unstimulated controls. Statistics were calculated using Wilcoxon ranksum test. *P < 0.05; **P < 0.01 ; ***P < 0.001 ; ns, not significant.

- Figure 3 shows the flowcytometry gating strategy of analysis to evaluate the memory CD8+ T cell responses in HRs following mRNA vaccine (BNT162b2).

■ Figure 4 shows that BNT162b2 mRNA vaccine promotes the differentiation of distinct memory CD8+ T cell subsets in HR, LR and EC cohorts. (A) Box plots showing the log of CM cell percentage on gated CD25 ⁺ CD8 ⁺ T cells from LR, HR and EC subjects at time 0, 6 weeks, 3 and 6 months after 48h of incubation with Spike, S_RBD_WT or S_RBD indicated variants and at 6 weeks and 6 months after 48h of incubation with S_RBD_B.1 .617.2, at time 0 and 6 months with S_RBD_B11529. (B) Box plots showing the logic of EM1 cell percentage on gated CD25 ⁺ CD8 ⁺ T cells from LR, HR and EC subjects at time 0, 6 weeks, 3 and 6 months after 48h of incubation with Spike, S_RBD_WT or S_RBD indicated variants and at 6 weeks and 6 months after 48h of incubation with S_RBD_B.1.617.2, at time 0 and 6 months with S_RBD_B11529. (C) Box plots showing the logic of EM3 cell percentage on gated CD25 ⁺ CD8 ⁺ T cells from LR, HR and EC subjects at time 0, 6 weeks, 3 and 6 months after 48h of incubation with Spike, S_RBD_WT or S_RBD indicated variants and at 6 weeks and 6 months after 48h of incubation with S_RBD_B.1.617.2, at time 0 and 6 months with S_RBD_B1 1529. Data are represented with background subtraction from paired unstimulated controls. Statistics were calculated using Wilcoxon rank-sum test. *P < 0.05; **P < 0.01 ; ***P < 0.001 .

- Figure 5 shows the efficacy of vaccine protection against COVID-19 caused by Delta and Omicron SARS-CoV-2 variants in HR, LR, and EC subjects.

(A) Graph showing the prevalence of the indicated SARS-CoV-2 variants from January 2021 to April 15 ^th 2022 in Italy, the COVID-19 cases are also shown. (B) Graph showing the percentage of COVID cases correlated with the indicated SARS- CoV-2 variant breakthrough in all cohorts, LRs, HRs, EC-LRs and EC-HRs subjects. Fisher’s exact test was calculated (p = 0,01 LRs vs HRs; p = 0,0006 EC-LRs vs EC- HRs).

EXAMPLE 1: Anti-spike IgG level and CD8 ⁺ T cell phenotype correlate with protection and efficacy against COVID-19 following mRNA vaccine and allow to distinguish low responders from high responders to the mRNA vaccine.

Materials and methods Cohort

In total 379 staff members of FPO-IRCSS Candiolo were recruited for the study. Table 1 shows the characteristics of the IRCCS Candiolo cohort.

Table 1

According to the IgG quantification, it was decided to focus the analysis on 43 subjects that were divided into 3 groups: 10 SARS-CoV-2- EC individuals, 15 SARS-CoV-2-N-LR and 18 SARS-CoV-2-N-HR donors to BNT162b2 vaccination, who were never exposed to SARS-CoV-2 infection. Blood samples were collected before the first vaccine dose (time 0), after 6 weeks, 3 and 6 months.

All donors signed informed consent forms approved by the Ethical Committee of the FPO /CRC Candiolo.

Sample processing

Blood samples were collected into heparin tubes via phlebotomy. Tubes were centrifuged at 800g for 5 minutes at 4°C to separate plasma that was used for serological analysis. Whole blood was diluted 1 :1 with PBS 1X (Sigma) and peripheral blood mononuclear cells (PBMCs) isolation was obtained by density gradient centrifugation using Lympholyte (Cederlane). Tubes were centrifuged at 2000 rpm for 30 minutes at room temperature and PBMCs were collected into new tubes. Cells were washed twice with PBS 1X, centrifuged at 1600 rpm for 10 minutes at 4°C, counted with the Burker chamber and cryopreserved in 10% DMSO in FBS.

IgG quantification

Chemiluminescence immunoassay (CLIA) was performed with TGS COVID- 19 Control Set (Technogenetics CVCLCSGM) and TGS COVID-19 IgG (CVCL100G) according to the manufacturer instructions to quantify IgG.

In vitro stimulation

Frozen PBMCs were thawed in complete medium (RPMI supplemented with 2,5% human serum from Aurogene, 1 % L-Glutamine, 1 % penicillin/streptomycin, 1 % non-essential amino acids, 1 % sodium pyruvate and 0,1 % [3-mercaptoethanol). Cells were centrifuged at 1600 rpm for 10 minutes at 4°C, counted with the Burker chamber and resuspended in complete medium to a density of 4x10 ⁶ cells/mL. PBMCs were cultured for either 14h or 48h with 15-mer peptide pools covering the complete sequence of the Spike protein of the wild-type SARS-CoV-2 (first strain from Wuhan) and with 15-mer peptide pools covering the complete sequence of the Spike RBD from wild-type, B.1.1.7, B.1.351 , P.1 , B.1.617.2, and B11529 SARS- CoV-2 (45) (JPT, 2 pg/peptide/mL for 16-48h incubation). Medium with DMSO was used as negative control. For each condition, triplicate wells containing 2x10 ⁵ cells in 200 pL were plated in 96-well round-bottom plates and incubated at 37°Cwith 5% CO2.

Extracellular staining and flow cytometry analysis

After 48h of incubation, PBMCs were stained for surface markers. Cells were washed with PBS 1X supplemented with 0.5% BSA and 2Mm EDTA (FACS buffer) and stained with Fixable Viability Stain 450 for 20 minutes at 4°C to discriminate viable from non-viable cells. Then PBMCs were labelled with the following antibodies for multiparametric flow cytometry: anti-CD3, anti-CD8, anti-CD25, anti- CD45RA, anti-CCR7 and anti-CD27. Surface staining was performed for 30 minutes at 4°C in FACS buffer. Then, cells were washed and fixed with 1 % PFA. Cells were then washed once and resuspended in FACS buffer for data acquisition. Flow cytometry data were acquired on a BD LSRFortessa X-20 instrument and analysed with FlowJo software.

Statistical analysis

Wilcoxon signed-rank (non-parametric, paired) and Wilcoxon rank-sum (non- parametric, unpaired) test were used for statistical analysis and p value was determined using Prism software (Graphpad Software, Inc.), or otherwise indicated in the text. * indicates P < 0.05, ** indicates P < 0.01 , *** indicates P < 0.001 , n.s. indicates not significant. N.D. indicates not determined.

Results

Distinct anti-spike IgG antibody titers in SARS-CoV-2-N and SARS-CoV- 2-EC subjects following mRNA vaccination.

A cohort of 379 health care workers at FPO-IRCSS Candiolo Cancer Institute in Italy was examined longitudinally at 5 time points. Workers were vaccinated with two doses of the BNT162b2 mRNA (Comirnaty) vaccine from Pfizer-BioNTech encoding the SARS-CoV-2 spike protein derived from the ancestral SARS-CoV-2 virus (2). Subjects were classified as: i) SARS-CoV-2-N (308 with no history of infection, always negative to SARS-CoV-2 RNA-PCR swabs); ii) seropositive (23, no history of infection but with low level of specific Ab against spike and/or nucleocapsid); iii) SARS-CoV-2-Ex-COVID-19 subjects (SARS-CoV-2-EC, 48 with a documented past infection event). Peripheral blood was collected before the first dose administration (time 0), and after 3 weeks, 6 weeks, 3 months and 6 months from the first inoculation. IgG antibodies for spike and SARS-CoV-2 proteins were quantified in plasma.

Based on the kinetics of anti-spike IgG levels, the SARS-CoV-2-ECs displayed higher baseline and a delayed decrease in IgG concentration during the follow-up period, as compared to SARS-CoV-2-N subjects (Fig. 1A). In particular, anti-spike IgG levels in SARS-CoV-2-N subjects significantly increased 3 and 6 weeks after vaccination and gradually decreased 3 and 6 months later (with median values of 0, 18.4, >160, 74.9 and 31 All/rnl, respectively), whereas they were maintained significantly high in SARS-CoV-2-ECs (median values of 19.9, >160, >160, >160 and 143.35 All/rnl at the same time points), except after 6 month, when they significantly felled down, although less dramatically than in SARS-CoV-2-N subjects (Fig. 1 A, right panel). Taken together, these results confirm that vaccination after SARS-CoV-2 infection promotes higher and more durable anti-spike IgG titers than in SARS-CoV-2-N subjects(29) (30, 31).

Of note, three months after vaccination, the results unraveled a bimodal distribution of anti-spike IgG values in SARS-CoV-2-N individuals (Fig. 1 B). Based on these results, the 308 SARS-CoV-2-N individuals were divided in 236 low responders and 62 high responders (LRs and HRs, respectively), by using the antimode value measured 3 months after vaccination (cut-off 135 All/ml) (Fig. 1 C, left panel). This profile was also observed in SARS-CoV-2-EC HRs and LRs (Fig. 1 C, right panel).

Altogether, these results indicate durable anti-spike IgG responses 6 months post-vaccination, with higher titers in SARS-CoV-2-ECs with pre-existing immunity to the virus. Moreover, based on the anti-spike IgG values measured 3 months after the first dose administration, long-term SARS-CoV-2-N HRs and LRs to the mRNA vaccination have been distinguished.

Long-term CD8 ⁺ T cell responses against Spike and S_RBD_WT and derived from variants vary among SARS-CoV-2-naive LR, HR and SARS-CoV- 2-EC subjects.

To verify if BNT162b2 mRNA vaccine can induce long-term poly-specific memory CD8 ⁺ T cell responses against the most widespread SARS-CoV-2 VOC (B.1 .1 .7, B.1 .351 , P1 , B.1 .617.2 and B.1 .1.529 (23), as compared with the response to S_RBD from WT virus, PBMCs isolated from SARS-CoV-2-N LRs, SARS-CoV- 2-N HRs, or SARS-CoV-2-ECs at time 0, 6 weeks, 3 and 6 months from vaccination, were stimulated or not with 15-mer peptide pools covering either total spike, S_RBD_WT or the multiple S_RBD variant sequences (Fig. 2) for 48h. Then, samples were analyzed for the expression of CD25 within CD3 ⁺ CD8 ⁺ T cells by multiparametric flowcytometry.

The frequencies of stimulated CD25 ⁺ CD8 ⁺ T cells was calculated as expression levels of the activation markers with background subtraction, namely the value of unstimulated control. The data indicate that, following BNT162b2 mRNA vaccination, the magnitude and durability of specific CD25 ⁺ CD8 ⁺ T cell responses reactive against spike, S_RBD_WT or derived from VOCs are increased in SARS- CoV-2-ECs and SARS-CoV-2-N HRs as compared to SARS-CoV-2-N LRs, especially 3 and 6 months after vaccination (Fig. 2).

Distinct memory CD8 ⁺ T cell subsets react against S_RBD variants in SARS-CoV-2-naive LR, HR and SARS-CoV-2-EC cohorts.

Since long-term memory immunity is a key determinant in fighting the SARS- CoV-2 pandemic, the level of heterogeneity and differentiation dynamics of memory CD8 ⁺ T cells (11) in SARS-CoV-2-naTve LR, HR and SARS-CoV-2-EC cohorts was evaluated (Fig. 3). As done before, PBMCs were stimulated or not in vitro for 48h with 15-mer peptide pools covering either total spike, the S_RBD_WT or derived from VOC (B.1.1.7, B.1.351 , P1 for all time points, and B.1.617.2 at 6 weeks and 6 months, at time 0 and 6 months with S_RBD_B11529). PBMCs cultured in the presence of IL-2 plus aCD3/aCD28-beads or hCMV derived-peptide pools were used as positive controls (not shown). Then, the expression of several differentiation markers was assessed via multiparametric flowcytometry on gated CD25 ⁺ CD8 ⁺ T cells, in order to distinguish the following subsets: CD45RA ⁺ CCR7 ⁺ CD27 ⁺ naive; CD45RA- CCR7 ⁺ CD27 ⁺ central memory (CM); CD45RA’ CCR7’ CD27 ⁺ (EM1 ); CD45RA- CCR7 ⁺ CD27’ (EM2); CD45RA’ CCR7’ CD27’ (EM3); and CD45RA ⁺ CCR7’ terminally differentiated effector memory (EMRA) subsets (Fig. 3 and 4). The Fig. 3 shows the gating strategy.

The percentages of CD25 ⁺ CD8 ⁺ CM cells specific to the entire spike, S_RBD_WT (Fig.4A), as well as the various S_RBD variants (B.1.1.7, B.1.351 , P1 , B.1.617.2, B.1.1.529) showed different behaviors: those specific to spike significantly increased especially after 3 months from vaccination only in SARS- CoV-2-N HRs, whereas this subset was poorly detectable after 3 and 6 months in SARS-CoV-2-N LRs. As opposite, CM cells increased at the 3rd and 6 ^th month in SARS-CoV-2-ECs (Fig.4A). Furthermore, the majority of S_RBD variant-specific CD25 ⁺ CD8 ⁺ EM1 cell frequencies increased over time in SARS-CoV-2-N HRs (Fig. 4B), whereas they did not change in SARS-CoV-2-N LRs along the course of the follow up, except in few subjects with spike, S_RBD_B.1.1.7 and S_RBD_B11529 (Fig.4B). The frequency of EM1 cells was found increased in SARS-CoV-2-EC samples collected at 6 weeks after vaccination and stimulated with spike, S_RBD_WT and S_RBD_P1 . Notably, the proportion of CD8 ⁺ EM3 cells significantly increased 6 months post-vaccination in SARS-CoV-2-N LRs, whereas it decreased over time in the SARS-CoV-2-EC cohort (Fig ,4C).

Taken together, these results indicate that CD8 ⁺ T cells activated by mRNA vaccination, undergo distinct memory differentiation programs in the three different cohorts. While EM1 and CM cell progression characterize the SARS-CoV-2-N HR and the SARS-CoV-2-EC cohorts, the EM3 subset is significantly enriched in SARS- CoV-2-N LRs.

Reduced vaccine efficacy in SARS-CoV-2-N LRs

Lastly, to validate if the different humoral responses and fate diversification of memory CD8 ⁺ T cells in the three cohorts correlate with COVID-19 protection, we measured the number of individuals who underwent infection with symptoms after immunization with two or three mRNA vaccine doses (Fig. 5A,B). The SARS-CoV- 2 epidemic in Italy has been characterized by four distinct SARS-CoV2 infection waves (www.ecdc.europa.eu), dominated by ancestral, followed by Alpha, Delta and Omicron variants, with Delta accounting for >90% sequence in December 2021 , and Omicron accounting for >90% since beginning of 2022 (Fig. 5A). Of note, 8 months after vaccination, 1.5% of the total individuals involved in the studies were infected with the Delta virus and developed mild COVID-19 (Fig. 5B), whereas between 10 and 15 months later, this frequency raised to 7.33% (Omicron wave, Fig. 5A,B). Noteworthy, all the individuals which developed COVID-19 after the second vaccine dose fell within SARS-CoV-2-N LRs (2,12 %, 5 subjects), while 9,1 % SARS-CoV-2-N LRs (21 subjects) developed the disease during the Omicron infection wave (and after the third dose). These results confirm the reduced humoral and cellular protection in SARS-CoV-2-N LR cohort (Fisher’s exact test p = 0,01 ). Interestingly, among SARS-CoV-2-ECs, only SARS-CoV-2-EC LRs (4/9; 44,4%) individuals developed COVID-19 (Fisher’s exact test p = 0,0006). Overall, these results indicate that the reduced RDB-binding Ab titers associated with impaired spike specific CD8 ⁺ cell responses correlate with increased susceptibility to COVID- 19 caused by Omicron infection, most probably because Omicron spike mutations occur also in regions targeted also by CD8 ⁺ T cells(46).

19.

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