ABSTRACT
BackgroundHomologous and heterologous SARS-CoV-2-vaccinations yield different spike protein-directed humoral and cellular immune responses. However, their interdependencies remain elusive. MethodsCOV-ADAPT is a prospective, observational cohort study of 417 healthcare workers who received homologous vaccination with Astra (ChAdOx1-S; AstraZeneca) or BNT (BNT162b2; Biontech/Pfizer) or heterologous vaccination with Astra/BNT. We assessed the humoral (anti-spike-RBD-IgG, neutralizing antibodies, antibody avidity) and cellular (spike-induced T cell interferon-{gamma} release) immune response in blood samples up to 2 weeks before (T1) and 2 to 12 weeks following secondary immunization (T2). FindingsInitial vaccination with Astra resulted in lower anti-spike-RBD-IgG responses compared to BNT (70{+/-}114 vs. 226{+/-}279 BAU/ml, p<0.01) at T1, whereas T cell activation did not differ significantly. Booster vaccination with BNT proved superior to Astra at T2 (anti-spike-RBD-IgG: Astra/BNT 2387{+/-}1627 and BNT/BNT 3202{+/-}2184 vs. Astra/Astra 413{+/-}461 BAU/ml, both p<0.001; spike-induced T cell interferon-{gamma} release: Astra/BNT 5069{+/-}6733 and BNT/BNT 4880{+/-}7570 vs. Astra/Astra 1152{+/-}2243 mIU/ml, both p<0.001). No significant differences were detected between BNT-boostered groups at T2. For Astra, we observed no booster effect on T cell activation. We found associations between anti-spike-RBD-IgG levels (Astra/BNT and BNT/BNT) and T cell responses (Astra/Astra and Astra/BNT) from T1 to T2. There were also links between levels of anti-spike-RBD-IgG and T cell at both time points (all groups combined). All regimes yielded neutralizing antibodies and increased antibody avidity at T2. InterpretationInterdependencies between humoral and cellular immune responses differ between common SARS-CoV-2 vaccination regimes. T cell activation is unlikely to compensate for poor humoral responses. FundingDeutsche Forschungsgemeinschaft (DFG), ER723/3-1 Research in contextO_ST_ABSEvidence before this studyC_ST_ABSWe searched Pubmed for papers published between 01/01/2019 and 14/05/2021 with the search terms "covid-19" combined with "vaccination" and "heterologous", excluding "BCG". Of the 41 papers found, none addressed the combination of ChAdOx1-S by AstraZeneca (Astra) and BNT162b2 by Biontech/Pfizer (BNT). After our study was initiated, the CombiVacS trial reported a significant booster effect when BNT was given after initial vaccination with Astra.1 The investigators of the CoCo trial subsequently published data on heterologous immunization in comparison to homologous Astra in a small population (n=87), with the heterologous immunization scheme showing a superior humoral and cellular immune response.2 Further studies investigated heterologous vaccinations with Astra and BNT as well as homologous Astra and BNT regimes and also found superior humoral and cellular immune responses in the heterologous regimes compared to homologous Astra, and comparable or slightly superior immune responses when compared to homologous BNT vaccination.3-6 The body of research covering the effects of heterologous immunization regimes has recently been aggregated in a systematic review.7 Added value of this studyTo our knowledge, this is the first study that evaluates the interdependencies of cellular and humoral immune responses following heterologous vaccination with Astra/BNT in a large group of individuals. Our data show strong correlations between humoral and cellular immune responses with the prime-boost combination Astra/BNT. The findings suggest that individuals with a robust initial response developed strong humoral and cellular immune responses after booster immunization. Implications of all the available evidenceOur study and the available data suggest that due to its superior capacity to elicit a humoral and cellular immune response, mRNA-based vaccines such as BNT should be chosen for booster vaccination rather than Astra. This seems to be particularly important in individuals whose immune response was poor after initial vaccination with Astra. We demonstrate here an association between humoral and cellular immune responses following vaccination. Our findings suggest that distinct differences between common COVID-19 vaccination regimes should be taken into account in population-based vaccine programs. The present data indicate that a poor humoral immune response is unlikely to be mitigated by a strong cellular immune response.
ABSTRACT
Effective therapeutics to inhibit the replication of SARS-CoV-2 in infected individuals are still under development. The nucleoside analogue N4-hydroxycytidine (NHC), also known as EIDD-1931, interferes with SARS-CoV-2 replication in cell culture. It is the active metabolite of the prodrug Molnupiravir (MK-4482), which is currently being evaluated for the treatment of COVID-19 in advanced clinical studies. Meanwhile, inhibitors of dihydroorotate dehydrogenase (DHODH), by reducing the cellular synthesis of pyrimidines, counteract virus replication and are also being clinically evaluated for COVID-19 therapy. Here we show that the combination of NHC and DHODH inhibitors such as teriflunomide, IMU-838/vidofludimus, and BAY2402234, strongly synergizes to inhibit SARS-CoV-2 replication. While single drug treatment only mildly impaired virus replication, combination treatments reduced virus yields by at least two orders of magnitude. We determined this by RT-PCR, TCID50, immunoblot and immunofluorescence assays in Vero E6 and Calu-3 cells infected with wildtype and the Alpha and Beta variants of SARS-CoV-2. We propose that the lack of available pyrimidine nucleotides upon DHODH inhibition increases the incorporation of NHC in nascent viral RNA, thus precluding the correct synthesis of the viral genome in subsequent rounds of replication, thereby inhibiting the production of replication competent virus particles. This concept was further supported by the rescue of replicating virus after addition of pyrimidine nucleosides to the media. Based on our results, we suggest combining these drug candidates, which are currently both tested in clinical studies, to counteract the replication of SARS-CoV-2, the progression of COVID-19, and the transmission of the disease within the population. SIGNIFICANCEO_LIThe strong synergy displayed by DHODH inhibitors and the active compound of Molnupiravir might enable lower concentrations of each drug to antagonize virus replication, with less toxicity. C_LIO_LIBoth Molnupiravir and DHODH inhibitors are currently being tested in advanced clinical trials or are FDA-approved for different purposes, raising the perspective of rapidly testing their combinatory efficacy in clinical studies. C_LIO_LIMolnupiravir is currently a promising candidate for treating early stages of COVID-19, under phase II/III clinical evaluation. However, like Remdesivir, it appears only moderately useful in treating severe COVID-19. Since the combination inhibits virus replication far more strongly, and since DHODH inhibitors may also suppress excessive immune responses, the combined clinical application bears the potential of alleviating the disease burden even at later stages. C_LI
ABSTRACT
The search for successful therapies of infections with the coronavirus SARS-CoV-2 is ongoing. We tested inhibition of host cell nucleotide synthesis as a promising strategy to decrease the replication of SARS-CoV-2-RNA, thus diminishing the formation of virus progeny. Methotrexate (MTX) is an established drug for cancer therapy and to induce immunosuppression. The drug inhibits dihydrofolate reductase and other enzymes required for the synthesis of nucleotides. Strikingly, the replication of SARS-CoV-2 was inhibited by MTX in therapeutic concentrations around 1 M, leading to more than 1000-fold reductions in virus progeny in Vero C1008 (Vero E6) as well as Calu-3 cells. Virus replication was more sensitive to equivalent concentrations of MTX than of the established antiviral agent remdesivir. MTX strongly diminished the synthesis of viral structural proteins and the amount of released virus RNA. Virus replication and protein synthesis were rescued by folinic acid (leucovorin) and also by inosine, indicating that purine depletion is the principal mechanism that allows MTX to reduce virus RNA synthesis. The combination of MTX with remdesivir led to synergistic impairment of virus replication, even at 300 nM MTX. The use of MTX in treating SARS-CoV-2 infections still awaits further evaluation regarding toxicity and efficacy in infected organisms, rather than cultured cells. Within the frame of these caveats, however, our results raise the perspective of a two-fold benefit from repurposing MTX for treating COVID-19. Firstly, its previously known ability to reduce aberrant inflammatory responses might dampen respiratory distress. In addition, its direct antiviral activity described here would limit the dissemination of the virus. SIGNIFICANCEO_LIMTX is one of the earliest cancer drugs to be developed, giving rise to seven decades of clinical experience. It is on the World Health Organizations List of Essential Medicines, can be administered orally or parenterally, and its costs are at single digit {euro} or $ amounts/day for standard treatment. In case of its successful further preclinical evaluation for treating SARS-CoV-2 infections, its repurposing to treat COVID-19 would thus be feasible, especially under low-resource conditions. C_LIO_LIAdditional drugs exist to interfere with the synthesis of nucleotides, e.g. additional folate antagonists, inhibitors of GMP synthetase, or inhibitors of dihydroorotate dehydrogenase (DHODH). Such inhibitors have been approved as drugs for different purposes and might represent further therapeutic options against infections with SARS-CoV-2 C_LIO_LIRemdesivir is currently the most established drug for treating COVID-19. Our results argue that MTX and remdesivir, even at moderate concentrations, can act in a synergistic fashion to repress virus replication to a considerably greater extent than either drug alone. C_LIO_LICOVID-19, in its severe forms, is characterized by pneumonia and acute respiratory distress syndrome, and additional organ involvements. These manifestations are not necessarily a direct consequence of virus replication and cytopathic effects, but rather a result of an uncontrolled inflammatory and immune response. Anti-inflammatory drugs such as glucocorticoids are thus being evaluated for treating COVID-19. However, this bears the risk of re-activating virus spread by suppressing a sufficient and specific immune response. In this situation, it is tempting to speculate that MTX might suppress both excessive inflammation as well as virus replication at the same time, thus limiting both the pathogenesis of pneumonia and also the spread of virus within a patient. C_LI
