Immunogenicity of bivalent omicron (BA.1) booster vaccination after different priming regimens in health-care workers in the Netherlands (SWITCH ON): results from the direct boost group of an open-label, multicentre, randomised controlled trial.

BACKGROUND: Bivalent mRNA-based COVID-19 vaccines encoding the ancestral and omicron spike (S) protein were developed as a countermeasure against antigenically distinct SARS-CoV-2 variants. We aimed to assess the (variant-specific) immunogenicity and reactogenicity of mRNA-based bivalent omicron (BA.1) vaccines in individuals who were primed with adenovirus-based or mRNA-based vaccines encoding the ancestral spike protein. METHODS: We analysed results of the direct boost group of the SWITCH ON study, an open-label, multicentre, randomised controlled trial. Health-care workers from four academic hospitals in the Netherlands aged 18-65 years who had completed a primary COVID-19 vaccination regimen and received one booster of an mRNA-based vaccine, given no later than 3 months previously, were eligible. Participants were randomly assigned (1:1) using computer software in block sizes of 16 and 24 to receive an omicron BA.1 bivalent booster straight away (direct boost group) or a bivalent omicron BA.5 booster, postponed for 90 days (postponed boost group), stratified by priming regimen. The BNT162b2 OMI BA.1 boost was given to participants younger than 45 years, and the mRNA-1273.214 boost was given to participants 45 years or older, as per Dutch guidelines. The direct boost group, whose results are presented here, were divided into four subgroups for analysis: (1) Ad26.COV2.S (Johnson & Johnson) prime and BNT162b2 OMI BA.1 (BioNTech-Pfizer) boost (Ad/P), (2) mRNA-based prime and BNT162b2 OMI BA.1 boost (mRNA/P), (3) Ad26.COV2.S prime and mRNA-1273.214 (Moderna) boost (Ad/M), and (4) mRNA-based prime and mRNA-1273.214 boost (mRNA/M). The primary outcome was fold change in S protein S1 subunit-specific IgG antibodies before and 28 days after booster vaccination. The primary outcome and safety were assessed in all participants except those who withdrew, had a SARS-CoV-2 breakthrough infection, or had a missing blood sample at day 0 or day 28. This trial is registered with ClinicalTrials.gov, NCT05471440. FINDINGS: Between Sept 2 and Oct 4, 2022, 219 (50%) of 434 eligible participants were randomly assigned to the direct boost group; 187 participants were included in the primary analyses; exclusions were mainly due to SARS-CoV-2 infection between days 0 and 28. From the 187 included participants, 138 (74%) were female and 49 (26%) were male. 42 (22%) of 187 participants received Ad/P and 44 (24%) mRNA/P (those aged <45 years), and 45 (24%) had received Ad/M and 56 (30%) mRNA/M (those aged ≥45 years). S1-specific binding antibody concentrations increased 7 days after bivalent booster vaccination and remained stable over 28 days in all four subgroups (geometric mean ratio [GMR] between day 0 and day 28 was 1·15 [95% CI 1·12-1·19] for the Ad/P group, 1·17 [1·14-1·20] for the mRNA/P group, 1·20 [1·17-1·23] for the Ad/M group, and 1·16 [1·13-1·19] for the mRNA/M group). We observed no significant difference in the GMR between the Ad/P and mRNA/P groups (p=0·51). The GMR appeared to be higher in the Ad/M group than in the mRNA/M group, but was not significant (p=0·073). Most side-effects were mild to moderate in severity and resolved within 48 h in most individuals. INTERPRETATION: Booster vaccination with mRNA-1273.214 or BNT162b2 OMI BA.1 in adult healthcare workers resulted in a rapid recall of humoral and cellular immune responses independent of the priming regimen. Monitoring of SARS-CoV-2 immunity at the population level, and simultaneously antigenic drift at the virus level, remains crucial to assess the necessity and timing of COVID-19 variant-specific booster vaccinations. FUNDING: The Netherlands Organization for Health Research and Development (ZonMw).

SARS-CoV-2 Spike-specific IFN-γ T-cell Response After COVID-19 Vaccination in Patients With Chronic Kidney Disease, on Dialysis, or Living With a Kidney Transplant.

Studies have shown that coronavirus disease 2019 (COVID-19) vaccination is associated with a lower humoral response in vulnerable kidney patients. Here, we investigated the T-cell response following COVID-19 vaccination in kidney patients compared with controls. Methods: Patients with chronic kidney disease (CKD) stage G4/5 [estimated glomerular filtration rate <30 mL/min/1.73 m2], on dialysis, or living with a kidney transplant and controls received 2 doses of the mRNA-1273 COVID-19 vaccine. Peripheral blood mononuclear cells were isolated at baseline and 28 d after the second vaccination. In 398 participants (50% of entire cohort; controls n = 95, CKD G4/5 n = 81, dialysis n = 78, kidney transplant recipients [KTRs] n = 144)' SARS-CoV-2-specific T cells were measured using an IFN-γ enzyme-linked immune absorbent spot assay. Results: A significantly lower SARS-CoV-2-specific T-cell response was observed after vaccination of patients on dialysis (54.5%) and KTRs (42.6%) in contrast to CDK G4/5 (70%) compared with controls (76%). The use of calcineurin inhibitors was associated with a low T-cell response in KTRs. In a subset of 20 KTRs, we observed waning of the cellular response 6 mo after the second vaccination, which was boosted to some extent after a third vaccination, although T-cell levels remained low. Conclusion: Our data suggest that vaccination is less effective in these patient groups, with humoral nonresponders also failing to mount an adequate cellular response, even after the third vaccination. Given the important role of T cells in protection against disease and cross-reactivity to SARS-CoV-2 variants, alternative vaccination strategies are urgently needed in these high-risk patient groups.

Re-polarization of immunosuppressive macrophages to tumor-cytotoxic macrophages by repurposed metabolic drugs.

M2-like tumor-associated macrophages promote tumor progression by establishing an immunosuppressive tumor microenvironment. The phenotype and activity of immunosuppressive macrophages are related to their mitochondrial metabolism. Thus, we studied if drugs targeting mitochondrial metabolic pathways can repolarize macrophages from M2 into an M1-like phenotype or can prevent M0-to-M2 polarization. The drugs selected are clinically approved or in clinical trials and target M2-specific metabolic pathways: fatty acid oxidation (Perhexiline and Trimetazidine), glutaminolysis (CB-839), PPAR activation (HX531), and mitochondrial electron transport chain (VLX-600). Murine bone marrow-derived macrophages were either polarized to M2 using IL-4 in the presence of the drugs or polarized first into M2 and then treated with the drugs in presence of IFN-γ for re-polarization. Targeting both fatty acid oxidation with Perhexiline or the electron transport chain with VLX-600 in the presence of IFN-γ, impaired mitochondrial basal, and maximal respiration and resulted in M2 to M1-like re-polarization (increased iNOS expression, NO production, IL-23, IL-27, and TNF-α secretion), similar to LPS+IFN-γ re-polarization. Moreover, drug-induced macrophage re-polarization resulted in a strong tumor-cytotoxic activity. Furthermore, the polarization of M0- to M2-like macrophages was impaired by CB-839, Trimetazidine, HX531, and Perhexiline, while Hx531 and Perhexiline also reduced MCP-1 secretion. Our results show that by targeting cell metabolism, macrophages could be re-polarized from M2- into an anti-tumoral M1-like phenotype and that M0-to-M2 polarization could be prevented. Overall, this study provides rational for the use of clinically applicable drugs to change an immunosuppressive tumor environment into a pro-inflammatory tumor environment that could support cancer immunotherapies.

First-in-Human Phase I Clinical Trial of an SFV-Based RNA Replicon Cancer Vaccine against HPV-Induced Cancers.

A first-in-human phase I trial of Vvax001, an alphavirus-based therapeutic cancer vaccine against human papillomavirus (HPV)-induced cancers was performed assessing immunological activity, safety, and tolerability. Vvax001 consists of replication-incompetent Semliki Forest virus replicon particles encoding HPV16-derived antigens E6 and E7. Twelve participants with a history of cervical intraepithelial neoplasia were included. Four cohorts of three participants were treated per dose level, ranging from 5 × 105 to 2.5 × 108 infectious particles per immunization. The participants received three immunizations with a 3-week interval. For immune monitoring, blood was drawn before immunization and 1 week after the second and third immunization. Immunization with Vvax001 was safe and well tolerated, with only mild injection site reactions, and resulted in both CD4+ and CD8+ T cell responses against E6 and E7 antigens. Even the lowest dose of 5 × 105 infectious particles elicited E6/E7-specific interferon (IFN)-γ responses in all three participants in this cohort. Overall, immunization resulted in positive vaccine-induced immune responses in 12 of 12 participants in one or more assays performed. In conclusion, Vvax001 was safe and induced immune responses in all participants. These data strongly support further clinical evaluation of Vvax001 as a therapeutic vaccine in patients with HPV-related malignancies.

Alphavirus-based hepatitis C virus therapeutic vaccines: can universal helper epitopes enhance HCV-specific cytotoxic T lymphocyte responses?

BACKGROUND: Antigen-specific T cell immune responses play a pivotal role in resolving acute and chronic hepatitis C virus (HCV) infections. Currently, no prophylactic or therapeutic vaccines against HCV are available. We previously demonstrated the preclinical potency of therapeutic HCV vaccines based on recombinant Semliki Forest virus (SFV) replicon particles. However, clinical trials do not always meet the high expectations of preclinical studies, thus, optimization of vaccine strategies is crucial. In efforts to further increase the frequency of HCV-specific immune responses in the candidate SFV-based vaccines, the authors assessed whether inclusion of three strong, so-called universal helper T cell epitopes, and an endoplasmic reticulum localization, and retention signal (collectively termed sigHELP-KDEL cassette) could enhance HCV-specific immune responses. METHODS: We included the sigHELP-KDEL cassette in two of the candidate SFV-based HCV vaccines, targeting NS3/4A and NS5A/B proteins. We characterized the new constructs in vitro for the expression and stability of the transgene-encoded proteins. Their immune efficacy with respect to HCV-specific immune responses in vivo was compared with the parental SFV vaccine expressing the corresponding HCV antigen. Further characterization of the functionality of the HCV-specific CD8+ T cells was assessed by surface and intracellular cytokine staining and flow cytometry analysis. RESULTS: Moderate, but significantly, enhanced frequencies of antigen-specific immune responses were achieved upon lower/suboptimal dosage immunization. In optimal dosage immunization, the inclusion of the cassette did not further increase the frequencies of HCV-specific CD8+ T cells when compared with the parental vaccines and the frequencies of effector and memory populations were identical. CONCLUSION: We hypothesize that the additional effect of the sigHELP-KDEL cassette in SFV-based vaccines depends on the immunogenicity, nature, and stability of the target antigen expressed by the vaccine.

Potent therapeutic efficacy of an alphavirus replicon DNA vaccine expressing human papilloma virus E6 and E7 antigens.

Cervical cancer develops as a result of infection with high-risk human papillomavirus (HPV) through persistent expression of early proteins E6 and E7. Our group pioneered a recombinant viral vector system based on Semliki Forest virus (SFV) for vaccination against cervical cancer. The most striking benefit of this alphavirus vector-based vaccine platform is its high potency. DNA vaccines on the other hand, have a major advantage with respect to ease of production. In this study, the benefits associated with both SFV-based vaccines and DNA vaccines were combined with the development of a DNA-launched RNA replicon (DREP) vaccine targeting cervical cancer. Using intradermal delivery followed by electroporation, we demonstrated that DREP encoding for E6,7 (DREP-E6,7) induced effective, therapeutic antitumor immunity. While immunizations with a conventional DNA vaccine did not prevent tumor outgrowth, immunization with a 200-fold lower equimolar dose of DREP (0.05 µg of DREP) resulted in approximately 85% of tumor-free mice. To overcome the safety concern of potential malignant transformation at the vaccination site, we evaluated the anti-tumor effect of a DREP vaccine encoding a shuffled version of E7 (DREP-E7sh). DREP-E7sh delayed tumor growth yet not to the same extent as DREP-E6,7. In addition, inclusion of a helper cassette and an ER targeting signal (sigHelp) did not significantly further enhance the suppression of tumor outgrowth in the long term, albeit exhibiting better tumor control early after immunization. Collectively, this study points towards the clinical evaluation of DREP encoding HPV antigens as a potent immunotherapy for patients with HPV16 (pre)-malignancies.

A rationally designed combined treatment with an alphavirus-based cancer vaccine, sunitinib and low-dose tumor irradiation completely blocks tumor development.

The clinical efficacy of therapeutic cancer vaccines remains limited. For effective immunotherapeutic responses in cancer patients, multimodal approaches capable of both inducing antitumor immune responses and bypassing tumor-mediated immune escape seem essential. Here, we report on a combination therapy comprising sunitinib (40 mg/kg), single low-dose (14 Gy) tumor irradiation and immunization with a therapeutic cancer vaccine based on a Semliki Forest virus vector encoding the oncoproteins E6 and E7 of human papillomavirus (SFVeE6,7). We previously demonstrated that either low-dose irradiation or sunitinib in single combination with SFVeE6,7 immunizations enhanced the intratumoral ratio of antitumor effector cells to myeloid-derived suppressor cells (MDSCs). On the basis of these results we designed a triple treatment combinatorial regimen. The trimodal sunitinib, low-dose irradiation and SFVeE6,7 immunization therapy resulted in stronger intratumoral MDSC depletion than sunitinib alone. Concomitantly, the highest levels of intratumoral E7-specific CD8+ T cells were attained after triple treatment. Approximately 75% of these cells were positive for the early activation marker CD69. The combination of sunitinib, low-dose tumor irradiation and SFVeE6,7 immunization dramatically changed the intratumoral immune compartment. Whereas control tumors contained 0.02 E7-specific CD8+ T cells per MDSC, triple treatment tumors contained more than 200 E7-specific CD8+ T cells per MDSC, a 10,000-fold increased ratio. As a result, the triple treatment strongly enhanced the immunotherapeutic antitumor effect, blocking tumor development altogether and leading to 100% tumor-free survival of tumor-bearing mice. This study demonstrates that this multimodal approach elicits superior antitumor effects and should be considered for clinical applications.

Alphavirus-based vaccines encoding nonstructural proteins of hepatitis C virus induce robust and protective T-cell responses.

An absolute prerequisite for a therapeutic vaccine against hepatitis C virus (HCV) infection is the potency to induce HCV-specific vigorous and broad-spectrum T-cell responses. Here, we generated three HCV vaccines based on a recombinant Semliki Forest virus (rSFV) vector expressing all- or a part of the conserved nonstructural proteins (nsPs) of HCV. We demonstrated that an rSFV vector was able to encode a transgene as large as 6.1 kb without affecting its vaccine immunogenicity. Prime-boost immunizations of mice with rSFV expressing all nsPs induced strong and long-lasting NS3-specific CD8(+) T-cell responses. The strength and functional heterogeneity of the T-cell response was similar to that induced with rSFV expressing only NS3/4A. Furthermore this leads to a significant growth delay and negative selection of HCV-expressing EL4 tumors in an in vivo mouse model. In general, as broad-spectrum T-cell responses are only seen in patients with resolved HCV infection, this rSFV-based vector, which expresses all nsPs, inducing robust T-cell activity has a potential for the treatment of HCV infections.

Prognostic role of indoleamine 2,3-dioxygenase in endometrial carcinoma.

OBJECTIVE: Indoleamine-2,3-dioxygenase (IDO) suppresses the function of T-lymphocytes and is an important immune escape mechanism for cancer. Therefore, it is to be expected that IDO influences prognosis of cancer patients. This study aimed to investigate the prognostic role of IDO expression in a large cohort of endometrial carcinoma (EC) patients. METHODS: A tissue microarray containing primary EC tissue of 355 patients treated in a single institution was used to evaluate IDO expression. Expression of IDO was associated with clinicopathological characteristics, survival and previously determined numbers of CD8(+) and Foxp3(+) T-lymphocytes. RESULTS: IDO(high) expression was associated with lower numbers of intratumoral CD8(+) T-lymphocytes (p=0.031). Next to well-known prognostic parameters, IDO(high) expression was independently associated with poor disease specific survival in the general cohort of EC patients (HR 2.62, 95% C.I. 1.48-4.66, p=0.001) and among patients with early stage EC (HR 3.06, 95% C.I. 1.10-8.54, p=0.032). CONCLUSION: Our results show that IDO expression is associated with poor survival. This provides evidence that further research into the use of IDO blocking agents in cancer treatment is valid where it might be a promising new therapeutic strategy.

Loss of HLA class I and mismatch repair protein expression in sporadic endometrioid endometrial carcinomas.

Tumor cells can escape from cytotoxic T-cell responses by downregulation of human leukocyte antigen (HLA) class I molecules expressed at the cell surface which has been associated with a deficient mismatch repair (MMR) system in colorectal carcinomas. Our study investigated the association between expression of MMR proteins and HLA class I in sporadic endometrioid endometrial carcinomas (EC). In a consecutively selected cohort of 486 EC patients, MMR proteins (MLH1, MSH2 and MSH6) and HLA class I (HLA-A, -B, -C or ß(2) m) were investigated by immunohistochemistry. Expression levels of MMR proteins and HLA class I were compared between low-grade and high-grade ECs. HLA class I expression was compared between tumors with loss (negative immunostaining of ≥1 MMR protein) and expression of MMR proteins. Associations between previously determined numbers of intratumoral CD8(+) T-lymphocytes and expression of MMR proteins and HLA class I and the influence on survival was determined. ECs with loss of MMR protein expression (33.5%) more frequently have loss of HLA-B/C (37.3%), compared to ECs with MMR protein expression (25.5%, p = 0.007). Patients with loss of MMR proteins have a worse disease-specific survival compared to patients with expression (p = 0.039). CD8(+) T-lymphocytes have a positive influence on disease-free and disease-specific survival in the total EC cohort but not in patients with loss of MMR protein expression. In conclusion, our results indicate that loss of MMR protein expression is related to selective downregulation of HLA class I which contributes to immune escape in EC with an abnormal MMR system.