Omicron (B.1.1.529) BA.1 or BA.2-related effects on immune responses in previously naïve versus imprinted individuals: immune imprinting as an advantage in the humoral immune response against novel variants.

Background: Immune imprinting is a phenomenon in which a person's immune system develops a specific immunological memory of the pathogen or vaccine due to a previous exposure. This memory basically leads to a faster and stronger immune response in a subsequent contact to the same pathogen or vaccine. However, what happens if the pathogen has changed considerably in the meantime due to mutations in the main target region of antibodies, as in the evolution of SARS-CoV-2 from the ancestral strain to B.1.1.529 (Omicron)? In this case, does immune imprinting also confer an advantage in repeated contact and does it lead to a stronger immune response? Methods: To clarify these questions, we investigated the effects of immune imprinting in the context of SARS-CoV-2 by comparing a group of previously infection-naïve versus imprinted study participants and determined differences in humoral and cellular immune responses during and after infection with strain SARS-CoV-2 B.1.1.529 BA.1 and BA.2, respectively. We used a commercial CLIA, immunoblots, IFN-γ ELISpots and a plaque-reduction neutralization test to generate a clear and comparable picture of the humoral and cellular immune response in the two study groups. Results: Imprinted participants developed significantly higher antibody titers and showed significantly stronger neutralization capacity against the ancestral strain, BA.1 and BA.5. The immune response of naïve study participants was narrower and related mainly to the receptor-binding domain, which resulted in a lower neutralization capacity against other strains including BA.5. Naïve study participants showed a significantly higher cellular immune response than the imprinted study group, indicating a higher antigenic challenge. The cellular immune response was directed against general structures of SARS-CoV-2 and not specifically against the receptor-binding domain. Conclusion: Viral variant infection elicits variant-specific antibodies and prior mRNA vaccination or infection with a previous SARS-CoV-2 variant imprints serological responses toward the ancestral strain rather than variant antigens. On the other hand, our study shows that the initially higher specific antibody titers due to former imprinting via vaccination or prior infection significantly increased the humoral immune response, and therefore outperformed the humoral immune response of naïve study participants.

Immune Responses Against SARS-CoV-2 WT and Delta Variant in Elderly BNT162b2 Vaccinees.

Background: Residents of nursing homes are one of the most vulnerable groups during the severe acute syndrome coronavirus 2 (SARS-CoV-2) pandemic. The aim of this study was to characterize cellular and humoral immune responses in >70-year-old participants before vaccination, after first and second vaccination with BNT162b2, in contrast to second-dose-vaccinated participants younger than 60 years. Methods: Peripheral blood mononuclear cells of 45 elderly and 40 younger vaccinees were analyzed by IFNγ ELISpot, specific immunoglobulin G antibody titers against SARS-CoV-2 spike protein, and neutralization abilities against SARS-CoV-2 wild-type (WT) and Delta variant (B.1.617.2). Results: Our results clearly demonstrate a significantly increased T cell response, IgG titers, and neutralization activities against SARS-CoV-2 WT and Delta between first and second vaccination with BNT162b2 in elderly vaccinees, thereby highlighting the importance of the second booster. Interestingly, similar cellular and humoral immune responses against SARS-CoV-2 WT and Delta were found after the second vaccine dose in the young and elderly groups. Conclusions: Our data demonstrate a full picture of cellular and humoral immune responses of BNT162b2-vaccinees in two age cohorts. In all vaccines, SARS-CoV-2 WT-specific antibodies with similar neutralizing activity were detected in all vaccinees. After the second vaccination, neutralization titers against SARS-CoV-2 Delta were impaired in both age groups compared with SARS-CoV-2 WT, thereby emphasizing the need for an additional booster to overcome rising variants of SARS-CoV-2.

Cumulative SARS-CoV-2 mutations and corresponding changes in immunity in an immunocompromised patient indicate viral evolution within the host.

Different scenarios explaining the emergence of novel variants of concern (VOC) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been reported, including their evolution in scarcely monitored populations, in animals as alternative hosts, or in immunocompromised individuals. Here we report SARS-CoV-2 immune escape mutations over a period of seven months in an immunocompromised patient with prolonged viral shedding. Signs of infection, viral shedding and mutation events are periodically analyzed using RT-PCR and next-generation sequencing based on naso-pharyngeal swabs, with the results complemented by immunological diagnostics to determine humoral and T cell immune responses. Throughout the infection course, 17 non-synonymous intra-host mutations are noted, with 15 (88.2%) having been previously described as prominent immune escape mutations (S:E484K, S:D950N, S:P681H, S:N501Y, S:del(9), N:S235F and S:H655Y) in VOCs. The high frequency of these non-synonymous mutations is consistent with multiple events of convergent evolution. Thus, our results suggest that specific mutations in the SARS-CoV-2 genome may represent positions with a fitness advantage, and may serve as targets in future vaccine and therapeutics development for COVID-19.

The mutational dynamics of the SARS-CoV-2 virus in serial passages in vitro.

Since its outbreak in 2019, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) keeps surprising the medical community by evolving diverse immune escape mutations in a rapid and effective manner. To gain deeper insight into mutation frequency and dynamics, we isolated ten ancestral strains of SARS-CoV-2 and performed consecutive serial incubation in ten replications in a suitable and common cell line and subsequently analysed them using RT-qPCR and whole genome sequencing. Along those lines we hoped to gain fundamental insights into the evolutionary capacity of SARS-CoV-2 in vitro. Our results identified a series of adaptive genetic changes, ranging from unique convergent substitutional mutations and hitherto undescribed insertions. The region coding for spike proved to be a mutational hotspot, evolving a number of mutational changes including the already known substitutions at positions S:484 and S:501. We discussed the evolution of all specific adaptations as well as possible reasons for the seemingly inhomogeneous potential of SARS-CoV-2 in the adaptation to cell culture. The combination of serial passage in vitro with whole genome sequencing uncovers the immense mutational potential of some SARS-CoV-2 strains. The observed genetic changes of SARS-CoV-2 in vitro could not be explained solely by selectively neutral mutations but possibly resulted from the action of directional selection accumulating favourable genetic changes in the evolving variants, along the path of increasing potency of the strain. Competition among a high number of quasi-species in the SARS-CoV-2 in vitro population gene pool may reinforce directional selection and boost the speed of evolutionary change.

Maintenance of neutralizing antibodies over ten months in convalescent SARS-CoV-2 afflicted patients.

Knowledge of the level and duration of protective immunity against SARS-CoV-2 after primary infection is of crucial importance for preventive approaches. Currently, there is a lack of evidence on the persistence of specific antibodies. We investigated the generation and maintenance of neutralizing antibodies of convalescent SARS-CoV-2-afflicted patients over a ten-month period post-primary infection using an immunofluorescence assay, a commercial chemiluminescent immunoassay and an in-house enzyme-linked neutralization assay. We present the successful application of an improved version of the plaque-reduction neutralization assay which can be analysed optometrically to simplify data interpretation. Based on the results of the enzyme-linked neutralization assay, neutralizing antibodies were maintained in 77.4% of convalescent individuals without relevant decay over ten months. Furthermore, a positive correlation between severity of infection and antibody titre was observed. In conclusion, SARS-CoV-2-afflicted individuals have been proven to be able to develop and maintain neutralizing antibodies over a period of ten months after primary infection. Findings suggest long-lasting presumably protective humoral immune responses after wild-type infection.

Adjunctive homeopathic treatment of hospitalized COVID-19 patients (COVIHOM): A retrospective case series.

BACKGROUND: and purpose. COVID-19 is a novel viral disease causing worldwide pandemia. The aim of this study was to describe the effect of adjunctive individualized homeopathic treatment delivered to hospitalized patients with confirmed symptomatic SARS-CoV-2 infection. PATIENT PRESENTATION: Thirteen patients with COVID-19 were admitted. Mean age was 73.4 ± 15.0 (SD) years. Twelve (92.3%) were speedily discharged without relevant sequelae after 14.4 ± 8.9 days. A single patient admitted in an advanced stage of septic disease died in hospital. A time-dependent improvement of relevant clinical symptoms was observed in the 12 surviving patients. Six (46.2%) were critically ill and treated in the intensive care unit (ICU). Mean stay at the ICU of the 5 surviving patients was 18.8 ± 6.8 days. In six patients (46.2%) gastrointestinal disorders accompanied COVID-19. CONCLUSION: The observations suggest that adjunctive homeopathic treatment may be helpful to treat patients with confirmed COVID-19 even in high - risk patients especially since there is no conventional treatment of COVID-19 available at present.

An in vitro model for assessment of SARS-CoV-2 infectivity by defining the correlation between virus isolation and quantitative PCR value: isolation success of SARS-CoV-2 from oropharyngeal swabs correlates negatively with Cq value.

BACKGROUND: At the beginning of the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), little was known about its actual rate of infectivity and any COVID-19 patient positive in laboratory testing was supposed to be highly infective and a public health risk factor. METHODS: One hundred oropharyngeal samples were obtained during routine work flow of testing symptomatic persons by quantitative polymerase chain reaction (qPCR) and were inoculated onto cell culture of VeroB4 cells to study the degree of infectivity of SARS-CoV-2 in vitro. Quantification by virus titration and an external standard using synthetic RNA gave the breaking point of infectivity in SARS-CoV-2 in vitro. RESULTS: A clear negative correlation (r = - 0.76; p < 0.05) could be asserted between the viral load in quantitative polymerase chain reaction (qPCR) and the probability of a successful isolation in serial isolation experiments of specific oropharyngeal samples positive in qPCR. Quantification by virus titration and an external standard using synthetic RNA indicate a Cq between 27 and 30 in E-gene screening PCR as a breaking point in vitro, where infectivity decreases significantly and isolations become less probable. CONCLUSIONS: This study showed that only the 21% of samples with the highest viral load were infectious enough to transmit the virus in vitro and determined that the dispersion rate in vitro is surprisingly close to those calculated in large retrospective epidemiological studies for SARS-CoV-2. This raises the question of whether this simple in vitro model is suitable to give first insights in dispersion characters of novel or neglected viral pathogens. The statement that SARS-CoV-2 needs at least 40,000 copies to reliably induce infection in vitro is an indication of its transmissibility in Public Health decisions. Applying quantitative PCR systems in diagnosis of SARS-CoV2 can distinguish between patients providing a high risk of transmission and those, where the risk of transmission is probably limited to close and long-lasting contacts.

SARS-CoV-2 mutations in MHC-I-restricted epitopes evade CD8+ T cell responses.

CD8+ T cell immunity to SARS-CoV-2 has been implicated in COVID-19 severity and virus control. Here, we identified nonsynonymous mutations in MHC-I-restricted CD8+ T cell epitopes after deep sequencing of 747 SARS-CoV-2 virus isolates. Mutant peptides exhibited diminished or abrogated MHC-I binding in a cell-free in vitro assay. Reduced MHC-I binding of mutant peptides was associated with decreased proliferation, IFN-γ production and cytotoxic activity of CD8+ T cells isolated from HLA-matched COVID-19 patients. Single cell RNA sequencing of ex vivo expanded, tetramer-sorted CD8+ T cells from COVID-19 patients further revealed qualitative differences in the transcriptional response to mutant peptides. Our findings highlight the capacity of SARS-CoV-2 to subvert CD8+ T cell surveillance through point mutations in MHC-I-restricted viral epitopes.

Home quarantine in COVID-19: A study on 50 consecutive patients in Austria.

A cohort of the first 50 COVID-19 patients in East Tyrol, a region in the southwest of Austria, were monitored in home quarantine. Specific viral ribonucleic acid was detected in throat swabs and stool samples. Analysis indicated a median virus shedding duration of 13 days; however, statistical outliers highlight the importance of consequent testing. This underlines the need of negative throat swabs prior to removing quarantine. We monitored the disease's characteristics via an in-house score called Corona Severity Index, in order to predict an aggravation of the disease. Special attention was paid to early symptoms, such as headache, which appeared to be significantly more common in younger patients (p=0.019). Anosmia and ageusia showed a predominance in female patients (p=0.028). Investigation revealed seven relapses and viral shedding fluctuation in four cases. A follow-up examination shed light on seroconversion which could be observed in 35 of 40 participants. This further clarifies the necessity of establishing discharge standards and follow-up management for COVID-19 patients.

Smell and Taste Disorders in COVID-19 Patients: Objective Testing and Magnetic Resonance Imaging in Five Cases.

Smell and taste disorders are acknowledged as characteristic symptoms for SARS-CoV-2 infection by now. These symptoms have been linked to a neuroinvasive course of disease. In this study, we investigated five consecutive COVID-19 patients with a prolonged course of dysosmia and dysgeusia. Those with objectifiable alteration in taste or smell were subjected to MRI with contrast agent to investigate possible involvement of the central nervous system. We found dysosmia and dysgeusia to be mostly objectifiable, but no evidence for neuroinvasiveness could be detected by MRI in the late stage of the disease. Alterations in taste and smell could be objectified in most patients. Nevertheless, no evidence for a neuroinvasive potential could be identified by MRI, at least in the late stage of disease. We encourage medical professionals to conduct specialized examinations and MRIs in the acute stage of disease, which guarantees an optimum patient care.