Objectives:We sought to identify determinants of vaccine response in people with multiple sclerosis (pwMS) receiving B-cell-depleting therapies. Methods:This was a prospective single-centre cohort study (ACTRN12623001249640). Peripheral blood samples were collected from pwMS receiving ocrelizumab (n = 38) before and after a third dose of COVID-19 mRNA vaccine. Immunogenicity was measured by T-cell IFN-γ ELISpot, antibody titres and live virus neutralisation. Humoral immunity was benchmarked against pwMS receiving natalizumab (n = 15), and against a correlate of real-world protection (50% reduction in incidence of infection). The peripheral immune phenotype was assessed by high-parameter flow cytometry and tested for association with vaccine response. Results:CD20+ T cells, natural killer (NK) cells and B cells were lower in pwMS receiving ocrelizumab, while CD27+CD38+ T-cell and CD8+ NK cell frequencies were elevated relative to natalizumab. Following a third dose, 51% of pwMS on ocrelizumab were seropositive for SARS-CoV-2 receptor-binding domain Ig, and 25% and 14% met the threshold for effective neutralisation of live ancestral and omicron BA.5 virus, respectively. B-cell frequency at the time of vaccination, but not time since ocrelizumab infusion, positively correlated with antibody response. Immunomodulatory CD56bright NK cells were negatively associated with antibody response. CD3-CD20+ B cells (% of lymphocytes) and CD56bright NK cells (% of NK cells) were associated with effective virus neutralisation in prior non-responders. Conclusion:Time since ocrelizumab infusion was not associated with protective vaccination. Evaluation of B-cell and CD56bright NK cell frequencies may provide a personalised strategy to stratify pwMS for vaccination and prophylaxis.

Zika virus (ZIKV) vaccine development has been hindered by the risk of antibody-dependent enhancement (ADE), particularly in dengue-endemic regions, where sub-neutralizing antibodies can exacerbate disease severity. T cell-based vaccines targeting non-structural (NS) antigens represent a safer alternative that bypasses this risk. Using immunocompetent BALB/c mice, we performed high-resolution in vivo mapping of ZIKV specific CD8⁺ and CD4⁺ T cell responses following ZIKVPRVABC59 infection, identifying high avidity, polyfunctional memory T cells targeting conserved NS1, NS3 and NS4 proteins. Guided by these data, we developed DNA vaccines encoding full-length NS3 and NS4 and evaluated their efficacy against ZIKV infection alone or combined with a validated construct encoding secreted NS1 (p-tpaNS1). NS3 and NS4 vaccination elicited robust cytotoxic and IFN-γ producing T cell responses, while co-administration with p-tpaNS1 significantly reduced peak serum viremia achieving earlier and stronger viral control. Although NS1 alone conferred strong protection, the multi-antigen formulation demonstrated additive benefits. This T cell-based vaccine approach, targeting conserved NS proteins, offers a scalable, thermostable platform with potential for safe deployment in childbearing women and resource-limited regions. Given NS protein conservation and cross-reactivity across flaviviruses, it also provides a promising foundation for next-generation pan-flavivirus vaccine development, although this remains to be directly tested.

Background: Failure to develop protective immunity in response to vaccination is common among kidney transplant recipients, rendering them susceptible to severe infection. Novel strategies are required. Here, we investigated the potential of mechanistic-target-of-rapamycin (mTOR) inhibitors to improve vaccine responses. Methods: Humoral and cellular responses to primary COVID-19 vaccination (ChAdOx1 or BNT162b2) were assessed for kidney transplant recipients receiving mTOR inhibitor-based (mTOR inhibitor, mycophenolate, prednisolone, N=15) and standard-of-care (tacrolimus, mycophenolate, prednisolone, N=40) immunosuppression, and healthy cohabitants (N=71), in a prospective observational study. Findings were validated and mechanisms explored in mice. Low/non-responding kidney transplant recipients receiving standard-of-care immunosuppression (N=54) were then randomized 1:1 to switch from mycophenolate to sirolimus, or remain on standard-of-care, for 4 weeks prior to receiving COVID-19 booster vaccination. Augmentation of immunity to COVID-19 was assessed as the primary outcome measure. Results: A 12-fold greater IFNγ-T cell response to primary vaccination was observed in kidney transplant recipients receiving mTOR inhibitor-based versus standard-of-care immunosuppression (520 vs 43 spot-forming units/10 6 cells, p < 0.001). A greater frequency of functional memory T cells in the mTOR inhibitor group was observed for both the CD4 + (0.20% vs. 0.05%, p < 0.001) and CD8 + (0.35% vs. 0.07%, p = 0.006) compartments by flow cytometry, and kidney transplant recipients receiving mTOR inhibitor-based immunosuppression produced greater frequencies of SARS-CoV-2-specific CD4 + T cells than healthy cohabitants (1.17% vs 0.48%, p = 0.03). In mice, sirolimus treatment enhanced both recall and de novo T cell responses to homologous and Omicron-specific booster vaccines. Switch from mycophenolate to sirolimus was well tolerated, however no significant difference was observed in the proportion of kidney transplant recipients in the intervention and control arms that achieved protective virus neutralization (10/25 [40%] vs 9/21 [43%] respectively, p = 0.85), nor in T cell response to vaccination (p = 0.89). Conclusions: mTOR inhibition was associated with improved T cell memory formation in kidney transplant recipients, however this effect was not reproduced by a short-term mycophenolate to sirolimus switch strategy.

Key PointsMechanistic target of rapamycin (mTOR) inhibitor-based immunosuppression was associated with an improved T-cell response to vaccination in kidney transplant recipients.Mice treated with an mTOR inhibitor exhibited improved T-cell responses to booster vaccination.Switching low and nonresponder kidney transplant recipients to an mTOR inhibitor did not improve T-cell response to a booster vaccination.BackgroundFailure to develop protective immunity in response to vaccination is common among kidney transplant recipients, rendering them susceptible to severe infection. Novel strategies are required. Here, we investigated the potential of mechanistic target of rapamycin (mTOR) inhibitors to improve vaccine responses.MethodsHumoral and cellular responses to primary coronavirus disease 2019 (COVID-19) vaccination (ChAdOx1 or BNT162b2) were assessed for kidney transplant recipients receiving mTOR inhibitor-based (mTOR inhibitor, mycophenolate, prednisolone, n=15) and standard-of-care (tacrolimus, mycophenolate, prednisolone, n=40) immunosuppression, and healthy cohabitants (n=71), in a prospective observational study. Findings were validated and mechanisms explored in mice. Low/nonresponding kidney transplant recipients receiving standard-of-care immunosuppression (N=54) were then randomized 1:1 to switch from mycophenolate to sirolimus or remain on standard of care for 4 weeks before receiving COVID-19 booster vaccination. Augmentation of immunity to COVID-19 was assessed as the primary outcome measure.ResultsA 12-fold greater IFN gamma T-cell response to primary vaccination was observed in kidney transplant recipients receiving mTOR inhibitor-based versus standard-of-care immunosuppression (520 versus 43 spot-forming units/106 cells, P < 0.001). A greater frequency of functional memory T cells in the mTOR inhibitor group was observed for both the CD4(+) (0.20% versus 0.05%, P < 0.001) and CD8(+) (0.35% versus 0.07%, P = 0.006) compartments by flow cytometry, and kidney transplant recipients receiving mTOR inhibitor-based immunosuppression produced greater frequencies of severe acute respiratory syndrome coronavirus 2-specific CD4(+) T cells than healthy cohabitants (1.17% versus 0.48%, P = 0.03). In mice, sirolimus treatment enhanced both recall and de novo T-cell responses to homologous and Omicron-specific booster vaccines. Switch from mycophenolate to sirolimus was well tolerated; however, no significant difference was observed in the proportion of kidney transplant recipients in the intervention and control arms that achieved protective virus neutralization (10/25 [40%] versus 9/21 [43%], respectively, P = 0.85) nor in T-cell response to vaccination (P = 0.89).ConclusionsmTOR inhibition was associated with improved T-cell memory formation in kidney transplant recipients; however, this effect was not reproduced by a short-term mycophenolate to sirolimus switch strategy.Clinical Trial registry name and registration number:Australian New Zealand Clinical Trials Registry, ACTRN12621001412820.

Antibody-dependent enhancement (ADE) of infection is a concern for flavivirus vaccine development. Poorly neutralising cross-reactive antibodies that target viral envelope can result in ADE. Vaccine strategies that harness T cell immunity should be explored. Nonstructural protein 1 (NS1) is a promising vaccine antigen that abrogates ADE risk. We developed a ZIKV NS1 DNA vaccine that is protective and immunogenic in mice. We evaluated NS1 vaccine in the pregnancy model of ZIKV infection showing protection of fetuses from IUGR, microcephaly, and brain damage. Vaccination of male IFNAR-/- mice prevented ZIKV-induced testicular damage and viral persistence. Protection was mediated by T cells. Vaccination of rhesus macaques with PharmaJet Tropis device showed that the vaccine is highly immunogenic and protective against ZIKV infection. Next, we developed NS1 mRNA vaccine and evaluated its protective efficacy. Inclusion of additional antigens can increase the breadth of T cell responses providing enhanced protection. To select additional antigens, we comprehensively evaluated in in vivo effector, early and late memory T cell responses after ZIKV infection. Complementing clinical data, we show that ZIKV NS3 and NS4 are the dominant T cell targets post-infection. Next we evaluated efficacy and immunogenicity of multivalent ZIKV NS1, NS3/4 mRNA vaccine. Our results have important implications for the development of multivalent ZIKV vaccines, that abrogate the risk of flavivirus ADE The Hospital Research Foundation Group (Australia), Medical research Future Fund (Australia) and National Foundation for Medical Research and Innovation (Australia) Vaccines and Immunotherapy (VAC)

Background and Objective People with multiple sclerosis (pwMS) receiving B cell-depleting therapies have impaired antibody responses to vaccination. In a proportion of individuals, repeat vaccination against COVID-19 leads to seroconversion. We sought to describe the immune phenotype of pwMS on ocrelizumab, and identify clinical and immunological determinants of an effective vaccine response. Methods This was a single-centre, prospective cohort study. Peripheral blood samples were collected from pwMS receiving ocrelizumab (n = 38) pre and post administration of a third dose of mRNA COVID-19 vaccine. Immunogenicity was measured by T cell IFNγ ELISpot, antibody titres, and live virus neutralisation. Humoral immunity was benchmarked against pwMS receiving natalizumab (n = 15), and against a correlate of real-world protection (50% reduction in incidence of infection) from SARS-CoV-2 ancestral and omicron BA.5 variants. The peripheral immune phenotype was comprehensively assessed by flow cytometry, and potential clinical and phenotypic determinants of response to vaccination identified. Results Immune cell populations relevant to disease and vaccine response were altered in pwMS receiving ocrelizumab versus natalizumab treatment, including depleted CD20-expressing B cell, T cell and NK cell populations, and elevated CD27+CD38+ T cell and ‘NK8’ cell frequencies. Following a third vaccine dose, 51% of pwMS on ocrelizumab were seropositive for SARS-CoV-2 receptor-binding-domain IgG, and 25% and 14% met the threshold for effective neutralisation of live SARS-CoV-2 ancestral and omicron BA.5 virus, respectively. B cell frequency at the time of vaccination, but not time since ocrelizumab infusion, was positively correlated with antibody response, while a strong negative correlation was observed between CD56bright NK cell frequency and antibody response in the ocrelizumab group. In this exploratory cohort, CD3−CD20+ B cells (% of lymphocytes; OR=3.92) and CD56bright NK cells (% of NK cells; OR=0.94) were predictive of an effective neutralising antibody response in second dose non-responders (AUC: 0.98). Discussion Ocrelizumab treatment was associated with an altered immune phenotype, including recently described T cell and NK populations with potential roles in disease pathogenesis. However, seroconversion was severely impaired by ocrelizumab, and less than half of those who seroconverted following a third vaccine dose demonstrated effective immunity against SARS-CoV-2 ancestral or omicron BA.5. B cell frequency was associated with an effective antibody response, while immunomodulatory CD56bright NK cells were identified as a potential negative determinant of response in those with inadequate B cell numbers. Immune phenotype rather than time since ocrelizumab infusion may help to stratify individuals for prophylaxis. ### Competing Interest Statement The authors have declared no competing interest. ### Clinical Trial ACTRN12623001249640 ### Funding Statement This work has received funding from The Hospital Research Foundation Group (outside of structured grant round) and the Health Services Charitable Gifts Board (HSCGB; project grant, 70-05-52-05-20). GBP and MJT received support from the Mary Overton Research Fellowship (HSCGB) and Jacquot Research Scholarship (Royal Australasian College of Physicians), respectively. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: This study was approved by the Central Adelaide Local Health Network Human Research Ethics Committee. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Phenotype and correlation data are available as a network (SIF) file, and raw values can be obtained by contacting G.B.P.

Inadequate immune response to vaccination is a long-standing problem faced by immunosuppressed kidney transplant recipients (KTRs), requiring novel strategies to improve vaccine efficacy. In this study, the potential of mechanistic target of rapamycin inhibitors (mTORi) to improve T cell responses to COVID-19 vaccination was investigated. Following primary vaccination with adenoviral (ChAdOx1) or mRNA (BNT162b2) COVID-19 vaccines, KTRs receiving rapamycin demonstrated T cell responses greater than those of healthy individuals, characterized by increased frequencies of vaccine-specific central memory, effector memory and TEMRA T cells, in both the CD4+ and CD8+ compartments. Relative to standard-of-care triple therapy, mTORi-based therapy was associated with a 12-fold greater functional T cell response to primary vaccination of KTRs. The use of rapamycin to augment T cell responses to COVID-19 booster (third dose) vaccination was next investigated in a randomized, controlled trial. Immunosuppression modification with rapamycin was feasible and well-tolerated, but did not improve vaccine-specific T cell responses in this cohort. To understand the parameters for effective use of rapamycin as a vaccine adjuvant, mice were treated with rapamycin before primary or booster vaccination with ancestral and/or Omicron COVID-19 vaccines. Supporting the findings from KTRs, significant enhancement of functional and stem-like memory T cell responses was observed when rapamycin was administered from the time of primary, rather than booster, vaccination. Collectively, a positive effect of mTOR inhibitors on vaccine-induced T cell immunity against COVID-19 in humans was demonstrated. One Sentence Summary Rapamycin use at the time of primary COVID-19 vaccination augments the formation of functional, vaccine-specific T cell memory in immunosuppressed kidney transplant recipients. ### Competing Interest Statement Alessandro Sette is a consultant for Gritstone Bio, Flow Pharma, Moderna, AstraZeneca, Qiagen, Fortress, Gilead, Sanofi, Merck, RiverVest, MedaCorp, Turnstone, NA Vaccine Institute, Emervax, Gerson Lehrman Group and Guggenheim. LJI has filed for patent protection for various aspects of T cell epitope and vaccine design work. All other authors declare no competing interests. ### Clinical Trial ACTRN12621000532808; ACTRN12621001412820 ### Clinical Protocols ### Funding Statement This work has received funding from The Hospital Research Foundation Group (outside of structured grant round) and the Health Services Charitable Gifts Board (HSCGB; project grant, 70-05-52-05-20), and was supported by NIH contract 75N93019C00065 (A.S, D.W). GBP and MJT received support from the Mary Overton Research Fellowship (HSCGB) and Jacquot Research Scholarship (Royal Australasian College of Physicians), respectively. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The Human Research Ethics Committee of the Central Adelaide Local Health Network gave ethical approval for human studies. The Animal Ethics Committee of the University of Adelaide gave ethical approval for animal studies. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present work are contained in the manuscript or available upon reasonable request to the authors.

Kidney transplant recipients (KTRs) are highly vulnerable to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and severe coronavirus disease 2019 (COVID-19). The 28-day case mortality for KTRs is 24%, and mounting evidence suggests poor immunogenicity and clinical effectiveness of vaccines in this group.1Phanish M. Ster I.C. Ghazanfar A. et al.Systematic review and meta-analysis of COVID-19 and kidney transplant recipients, the South West London Kidney Transplant Network experience.Kidney Int Rep. 2021; 6: 574-585Google Scholar,2Callaghan C.J. Mumford L. Curtis R.M. et al.Real-world effectiveness of the Pfizer-BioNTech BNT162b2 and Oxford-AstraZeneca ChAdOx1-S vaccines against SARS-CoV-2 in solid organ and islet transplant recipients.Transplantation. 2022; 106: 436-446Google Scholar Priority COVID-19 vaccination and booster dosing of close household contacts, that is, ring vaccination, has been suggested as an additional layer of protection for immunocompromised individuals.3Prendecki M. Thomson T. Clarke C.L. et al.Immunological responses to SARS-CoV-2 vaccines in kidney transplant recipients.Lancet. 2021; 398: 1482-1484Google Scholar To address this concept, KTRs and their healthy cohabitants were concurrently vaccinated, and their immunity to SARS-CoV-2 was compared in a registered observational clinical trial (ACTRN12621000532808). Forty-six kidney-alone transplant recipients from a South Australian transplant unit receiving immunosuppression with a calcineurin inhibitor, antimetabolite (mycophenolate mofetil or azathioprine), and steroid were included (Table 1). Each patient was paired with a healthy domestic cohabitant of similar age. Participants received 2 doses of either the ChAdOx1 (Oxford-AstraZeneca; n = 26 pairs) or the BNT162b2 (Pfizer-BioNTech; n = 20 pairs) vaccine as per Australian Government recommendations. Transplant recipients were predominantly male (n = 31 [67%]) and first-graft recipients (n = 40 [87%]), aged 60.8 ± 12.5 years, with transplant age 6.9 ± 7.2 years (see Table 1).Table 1Participant characteristicsCharacteristicTransplant (n = 46)Cohabitants (n = 46)Age, yr60.8 ± 12.559.2 ± 12.5SexF: 15; M: 31F: 31; M: 15VaccineChAdOx1: 26BNT162b2: 20ChAdOx1: 25BNT162b2: 21Cause of kidney failureGlomerulonephritis: 17 (37%)Other: 12 (26%)Polycystic kidney disease: 11 (24%)Diabetes mellitus: 3 (6.5%)Hypertension/renovascular: 2 (4.5%)Unknown: 1 (2%)NAAge of the transplanted kidney, yrRange: 0.2–34.6Mean: 6.9 ± 7.2Median 5.0NAGraft numberFirst graft: 40Second graft: 6NAGraft functioneGFR: 54.8 ± 18.5 ml/min/m2NAeGFR, estimated glomerular filtration rate; F, female; M, male; NA, not applicable. Open table in a new tab eGFR, estimated glomerular filtration rate; F, female; M, male; NA, not applicable. Circulating anti–receptor-binding domain (RBD) IgG antibodies (Elecsys, Roche) and serological neutralization of live SARS-CoV-2 were measured after 2 doses (median 21 days; interquartile range 21–24 days) of the BNT162b2 or ChAdOx1 vaccine as correlates of protection from symptomatic COVID-19.4Khoury D.S. Cromer D. Reynaldi A. et al.Neutralizing antibody levels are highly predictive of immune protection from symptomatic SARS-CoV-2 infection.Nat Med. 2021; 27: 1205-1211Google Scholar The study was further designed to enable cohabitants to serve as a healthy control group against which the broader anti-spike IgG antibody and T-cell responses of KTRs over time were compared. There was no COVID-19 community transmission in South Australia at the time of the study, and SARS-CoV-2 infection was excluded by serology in all participants by an accredited pathology service (SA Pathology; SARS-CoV-2 nucleocapsid IgG, Elecsys, Roche). After the first vaccine dose, anti-spike IgG (>2 SDs above baseline) was detected in only 27.0% of transplant recipients and 83.3% of cohabitants. After 2 vaccine doses, 100% of cohabitants had detectable titers of anti-spike IgG compared with only 44% of transplant recipients (Figure 1a). After the full course of COVID-19 immunization (2 doses), KTRs had a median anti-spike IgG titer >1000-fold lower than that of healthy cohabitants (area under the curve 0.83 versus 1454; Figure 1a). Where anti-spike IgG titer reflects the magnitude of antibody response to the vaccine, anti-RBD IgG and live virus neutralization are measures of functional immunity to SARS-CoV-2. Seroconversion of anti-RBD IgG (detection limit 0.4 U/ml) and serological neutralization were both achieved by 100% of cohabitants. In contrast, 10.9% of KTRs showed serum neutralizing activity and 32.6% had detectable titers of anti-RBD IgG (Figure 1b and c). To evaluate the level of protection afforded these groups, an anti-RBD IgG titer of 100 U/ml and serological neutralization of 40 were used as benchmarks for a protective vaccine response on the basis of similar studies and accepted thresholds for influenza hemagglutination assays.4Khoury D.S. Cromer D. Reynaldi A. et al.Neutralizing antibody levels are highly predictive of immune protection from symptomatic SARS-CoV-2 infection.Nat Med. 2021; 27: 1205-1211Google Scholar, 5Cox R. Correlates of protection to influenza virus, where do we go from here?.Hum Vacc Immunother. 2013; 9: 405-408Google Scholar, 6Hall V.G. Ferreira V.H. Ku T. et al.Randomized trial of a third dose of mRNA-1273 vaccine in transplant recipients.N Engl J Med. 2021; 385: 1244-1246Google Scholar All cohabitants met these thresholds, achieving anti-RBD IgG titers >100 U/ml and 50% live virus neutralization at serum dilutions of ≥1/40. Evidence of protective immunity was rarely achieved by transplant recipients, with 4.3% exceeding 100 U/ml of anti-RBD IgG and 8.7% achieving serological neutralization of ≥40. Antiviral T-cell responses are important in viral clearance and for minimizing the severity of COVID-19, particularly in the absence of an effective neutralizing antibody response.7Bange E.M. Han N.A. Wileyto P. et al.CD8+ T cells contribute to survival in patients with COVID-19 and hematologic cancer.Nat Med. 2021; 27: 1280-1289Google Scholar Conserved SARS-CoV-2 epitopes recognized by T cells may also provide cross-protection against viral variants that evade antibody neutralization.8Le Bert N. Tan A.T. Kunasegaran K. et al.SARS-CoV-2-specific T cell immunity in cases of COVID-19 and SARS, and uninfected controls.Nature. 2020; 584: 457-462Google Scholar Therefore, we evaluated the magnitude of spike-specific T-cell responses (reported by frequency of antigen-induced interferon-γ secretion) in KTRs and cohabitants by enzyme-linked immunosorbent spot (ELISpot) assay before vaccination and after 2 vaccine doses (Figure 1d). In line with previous reports (e.g., Anft et al.9Anft M. Blazquez-Navarro A. Stervbo U. et al.Detection of pre-existing SARS-CoV-2-reactive T cells in unexposed renal transplant patients.J Nephrol. 2021; 34: 1025-1037Google Scholar), preexisting T-cell immunity to SARS-CoV-2 spike was detected in both KTRs and cohabitants (35% and 60%, respectively). T-cell responses were increased upon vaccination in 49% of transplant recipients compared with 93% of cohabitants. The median increase from baseline in T-cell response of transplant recipients was 12.6-fold lower than that of cohabitants (22 spot-forming units per 106 cells versus 278 spot-forming units per 106 cells). This study provides the first assessment of vaccine-induced SARS-CoV-2 immunity in KTRs compared with synchronously vaccinated controls. KTRs were found to have a profoundly impaired capacity to generate specific IgG after 1 and 2 vaccine doses. Recent studies have also observed reduced T-cell responses in KTRs3Prendecki M. Thomson T. Clarke C.L. et al.Immunological responses to SARS-CoV-2 vaccines in kidney transplant recipients.Lancet. 2021; 398: 1482-1484Google Scholar; however, significant preexisting T-cell reactivity has confounded interpretation. Subtracting baseline T-cell reactivity allowed us to accurately measure the vaccine-induced cellular response and revealed a significant impairment in the capacity of KTRs to form antiviral T-cell immunity. The majority of SARS-CoV-2 transmission occurs between household contacts,S1 and this is likely to be exaggerated in transplant recipients because of practiced caution at avoiding infection in the community. Although epidemiological studies will be important for assessing the effect of vaccination on virus transmission to immunocompromised individuals, live virus neutralization is the current best in vitro correlate of protection from SARS-CoV-2 infection. In the study cohort, all cohabitants met the threshold for effective serological neutralization, as well as that set for anti-RBD IgG titer. By contrast, only 8.7% of KTRs met at least one of the thresholds. The choice of vaccine did not significantly influence the immune response in KTRs; however, superior IgG titers and serological neutralization were observed in household controls who received BNT162b2 (Supplementary Figures S1–S4). In light of recent findings of poor real-world effectiveness of the BNT162b2 and ChAdOx1 vaccines in solid organ transplant recipients, these data provide strong support for ring vaccination of cohabitants to reduce the risk of SARS-CoV-2 infection.2Callaghan C.J. Mumford L. Curtis R.M. et al.Real-world effectiveness of the Pfizer-BioNTech BNT162b2 and Oxford-AstraZeneca ChAdOx1-S vaccines against SARS-CoV-2 in solid organ and islet transplant recipients.Transplantation. 2022; 106: 436-446Google Scholar The effectiveness of ring vaccination in the real world is likely to depend on additional factors, including vaccination status of the whole household, including children as viable vaccination strategies for children are developed, and the emergence and spread of immune-evasive SARS-CoV-2 variants. With no forthcoming strategy to enhance vaccine immunogenicity in KTRs, and efforts underway to develop booster vaccines against the Omicron variant, priority booster vaccination of household contacts should be the preferred vaccination strategy to protect immunocompromised transplant recipients. All the authors declared no competing interests. This study was funded by project grants from The Hospital Research Foundation Group , Adelaide, Australia. Download .pdf (1.19 MB) Help with pdf files Supplementary File (PDF)

Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly infectious respiratory virus which is responsible for the coronavirus disease 2019 (COVID-19) pandemic. It is increasingly clear that recovered individuals, even those who had mild COVID-19, can suffer from persistent symptoms for many months after infection, a condition referred to as “long COVID”, post-acute sequelae of COVID-19 (PASC), post-acute COVID-19 syndrome, or post COVID-19 condition. However, despite the plethora of research on COVID-19, relatively little is known about the molecular underpinnings of these long-term effects. Methods We have undertaken an integrated analysis of immune responses in blood at a transcriptional, cellular, and serological level at 12, 16, and 24 weeks post-infection (wpi) in 69 patients recovering from mild, moderate, severe, or critical COVID-19 in comparison to healthy uninfected controls. Twenty-one of these patients were referred to a long COVID clinic and > 50% reported ongoing symptoms more than 6 months post-infection. Results Anti-Spike and anti-RBD IgG responses were largely stable up to 24 wpi and correlated with disease severity. Deep immunophenotyping revealed significant differences in multiple innate (NK cells, LD neutrophils, CXCR3+ monocytes) and adaptive immune populations (T helper, T follicular helper, and regulatory T cells) in convalescent individuals compared to healthy controls, which were most strongly evident at 12 and 16 wpi. RNA sequencing revealed significant perturbations to gene expression in COVID-19 convalescents until at least 6 months post-infection. We also uncovered significant differences in the transcriptome at 24 wpi of convalescents who were referred to a long COVID clinic compared to those who were not. Conclusions Variation in the rate of recovery from infection at a cellular and transcriptional level may explain the persistence of symptoms associated with long COVID in some individuals.

Additional file 13. Correlation network at 16-weeks post infection in Simple Interaction Format. The first column is a source node, second the spearman correlation coefficient and third the target node. File related to Fig. 6B.

Coronavirus disease 2019 (COVID-19) convalescents living in regions with low vaccination rates rely on post-infection immunity for protection against re-infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We evaluate humoral and T cell immunity against five variants of concern (VOCs) in mild-COVID-19 convalescents at 12 months after infection with ancestral virus. In this cohort, ancestral, receptor-binding domain (RBD)-specific antibody and circulating memory B cell levels are conserved in most individuals, and yet serum neutralization against live B.1.1.529 (Omicron) is completely abrogated and significantly reduced for other VOCs. Likewise, ancestral SARS-CoV-2-specific memory T cell frequencies are maintained in >50% of convalescents, but the cytokine response in these cells to mutated spike epitopes corresponding to B.1.1.529 and B.1.351 (Beta) VOCs were impaired. These results indicate that increased antigen variability in VOCs impairs humoral and spike-specific T cell immunity post-infection, strongly suggesting that COVID-19 convalescents are vulnerable and at risk of re-infection with VOCs, thus stressing the importance of vaccination programs.

Increasing evidence suggests immune dysregulation in individuals recovering from SARS- CoV-2 infection. We have undertaken an integrated analysis of immune responses at a transcriptional, cellular, and serological level at 12, 16, and 24 weeks post-infection (wpi) in 69 individuals recovering from mild, moderate, severe, or critical COVID-19. Anti-Spike and anti-RBD IgG responses were largely stable up to 24wpi and correlated with disease severity. Deep immunophenotyping revealed significant differences in multiple innate (NK cells, LD neutrophils, CXCR3 + monocytes) and adaptive immune populations (T helper, T follicular helper and regulatory T cells) in COVID-19 convalescents compared to healthy controls, which were most strongly evident at 12 and 16wpi. RNA sequencing suggested ongoing immune and metabolic dysregulation in convalescents months after infection. Variation in the rate of recovery from infection at a cellular and transcriptional level may explain the persistence of symptoms associated with long COVID in some individuals.

Background: Duration and quality of immunity to SARS-CoV-2 have significant implications for the management of COVID-19 pandemic. Here, we present a comprehensive set of immunological data from a cohort of individuals (n=43), 12 months after mild COVID-19 disease and in the absence of virus re-exposure.Methods: Serum and PBMC were collected from mild-COVID-19 convalescents 12 months after the COVID-19 positive PCR (n=43) and from healthy SARS-CoV-2-seronegative controls (n=15). Serum titers of SARS-CoV-2-specific immunoglobulins were quantified by ELISA and virus neutralisation activity was assessed using SARS-CoV-2-Spike pseudovirus particles. Frequencies of Spike and RBD-specific memory B cells were quantified by flow cytometry. Magnitude of memory T cell responses was quantified and phenotyped with an activation-induced marker assay.Findings: In the absence of re-exposure to SARS-CoV-2 Spike- and RBD-specific antibodies were present in 90% of COVID-19 convalescents 12 months post-infection. RBD-specific IgG + memory B cells were maintained in 88.9% of patients, while 62% of patients had serum neutralising activity. Functionally mature memory CD4 + and CD8 + T cells were maintained at frequencies previously reported for earlier time points post-COVID-19, indicating substantial maintenance of durable T cell responses. Interpretations: Immunity to SARS-CoV-2 persists for 12 months in mild COVID-19 convalescent patients that retain high Spike-specific antibody titres, virus neutralisation capacity and circulating RBD-specific memory B cells. Significantly, T cell immunity remained stable 12 months post-infection. This study offers vital information on the duration of natural COVID-19 immunity and its potential protective effect against SARS-CoV-2 reinfection and clinical disease, with clear implications for the ongoing management of the global pandemic. Funding Statement: This work was funded by project grants from The Hospital Research Foundation and Women's and Children's Foundation, Adelaide, Australia. This work has been supported by NIH contract 75N9301900065 (A.S, D.W).Declaration of Interests: A.S. is currently a consultant for Gritstone, Flow Pharma, Arcturus, Epitogenesis, Oxfordimmunotech, Caprion and Avalia. LJI has filed for patent protection for various aspects of T cell epitope and vaccine design work.Authors PGV, CMH, MGM, AELY, HB, ZAM, ZAD, AA, DA, JG, CF, SO, EMM, DJL, GM, EJG, BAJR, DS, CKL, MRB, DW, RAB, SCB and BGB declare no conflict of interest.Ethics Approval Statement: Study protocols were approved by the Central Adelaide Clinical Human Research Ethics Committee (#13050) and the Women's and Children's Health Network Human research ethics (protocol HREC/19/WCHN/65), Adelaide, Australia.

Background The duration and magnitude of SARS-CoV-2 immunity after infection, especially with regard to the emergence of new variants of concern (VoC), remains unclear. Here, immune memory to primary infection and immunity to VoC was assessed in mild-COVID-19 convalescents one year after infection and in the absence of viral re-exposure or COVID-19 vaccination. Methods Serum and PBMC were collected from mild-COVID-19 convalescents at ~6 and 12 months after a COVID-19 positive PCR (n=43) and from healthy SARS-CoV-2-seronegative controls (n=15-40). Serum titers of RBD and Spike-specific Ig were quantified by ELISA. Virus neutralisation was assessed against homologous, pseudotyped virus and homologous and VoC live viruses. Frequencies of Spike and RBD-specific memory B cells were quantified by flow cytometry. Magnitude of memory T cell responses was quantified and phenotyped by activation-induced marker assay, while T cell functionality was assessed by intracellular cytokine staining using peptides specific to homologous Spike virus antigen and four VoC Spike antigens. Findings At 12 months after mild-COVID-19, >90% of convalescents remained seropositive for RBD-IgG and 88.9% had circulating RBD-specific memory B cells. Despite this, only 51.2% convalescents had serum neutralising activity against homologous live-SARS-CoV-2 virus, which decreased to 44.2% when tested against live B.1.1.7, 4.6% against B.1.351, 11.6% against P.1 and 16.2%, against B.1.617.2 VoC. Spike and non-Spike-specific T cells were detected in >50% of convalescents with frequency values higher for Spike antigen (95% CI, 0.29-0.68% in CD4+ and 0.11-0.35% in CD8+ T cells), compared to non-Spike antigens. Despite the high prevalence and maintenance of Spike-specific T cells in Spike 'high-responder' convalescents at 12 months, T cell functionality, measured by cytokine expression after stimulation with Spike epitopes corresponding to VoC was severely affected. Interpretations SARS-CoV-2 immunity is retained in a significant proportion of mild COVID-19 convalescents 12 months post-infection in the absence of re-exposure to the virus. Despite this, changes in the amino acid sequence of the Spike antigen that are present in current VoC result in virus evasion of neutralising antibodies, as well as evasion of functional T cell responses.