Direct comparison of SARS-CoV-2 variant specific neutralizing antibodies in human and hamster sera.

Antigenic characterization of newly emerging SARS-CoV-2 variants is important to assess their immune escape and judge the need for future vaccine updates. To bridge data obtained from animal sera with human sera, we analyzed neutralizing antibody titers in human and hamster single infection sera in a highly controlled setting using the same authentic virus neutralization assay performed in one laboratory. Using a Bayesian framework, we found that titer fold changes in hamster sera corresponded well to human sera and that hamster sera generally exhibited higher reactivity.

Comparative analysis of SARS-CoV-2 neutralization titers reveals consistency between human and animal model serum and across assays.

The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires ongoing monitoring to judge the ability of newly arising variants to escape the immune response. A surveillance system necessitates an understanding of differences in neutralization titers measured in different assays and using human and animal serum samples. We compared 18 datasets generated using human, hamster, and mouse serum and six different neutralization assays. Datasets using animal model serum samples showed higher titer magnitudes than datasets using human serum samples in this comparison. Fold change in neutralization of variants compared to ancestral SARS-CoV-2, immunodominance patterns, and antigenic maps were similar among serum samples and assays. Most assays yielded consistent results, except for differences in fold change in cytopathic effect assays. Hamster serum samples were a consistent surrogate for human first-infection serum samples. These results inform the transition of surveillance of SARS-CoV-2 antigenic variation from dependence on human first-infection serum samples to the utilization of serum samples from animal models.

Detection of infectious SARS-CoV-2 in ocular samples is linked to viral load in the nasopharynx.

Introduction: SARS-CoV-2 is known to infect respiratory tissue cells. However, less is known about infection of ocular tissue and potential infectivity of lacrimal fluid. With this study, we want to compare viral loads in eye and nasopharyngeal swabs and analyze these for infectious virus. Methods: Between May 2020 and April 2021 ocular and nasopharyngeal swabs were collected from 28 SARS-CoV-2 infected patients treated on the corona virus disease 2019 (COVID-19)-ward of the University Hospital of Innsbruck, Austria. Samples with PCR detectable SARS-CoV-2 were analyzed via whole genome sequencing and an attempt was made to isolate infectious virus. Results: At the time point of sample collection, 22 individuals were still PCR positive in nasopharyngeal samples and in 6 of these patients one or both ocular samples were additionally positive. CT-values in eyes were generally higher compared to corresponding nasopharyngeal samples and we observed a tendency for lower CT-values, i.e. increased viral load, in nasopharyngeal swabs of individuals with at least one infected eye, compared to those where ocular samples were PCR negative. Ocular and nasopharyngeal sequences from the same patient were assigned to the same variant, either the D614G or the Alpha variant. Infectious virus was successfully isolated from 9 nasopharyngeal swabs, however only from one of the seven PCR positive ocular samples. Conclusion: We could detect SARS-CoV-2 in eyes of some of the infected patients albeit at lower levels compared to nasopharyngeal swabs. However, our results also indicate that lacrimal fluid might be infectious in patients with high viral load.

Durability of Cross-Neutralizing Antibodies 5.5 Months After Bivalent Coronavirus Disease 2019 Vaccine Booster.

We analyzed neutralizing antibodies in samples from ancestral + BA.1 and ancestral + BA.4/5 boosted individuals, collected around 5.5 months after booster. Titers of neutralizing antibodies generally decreased compared to a time point early after the bivalent booster immunization. This was more pronounced for individuals without infection history and for recently emerged Omicron variants.

Potency and durability of T and B cell immune responses after homologous and heterologous vector delivery of a trimer-stabilized, membrane-displayed HIV-1 clade ConC Env protein.

Introduction: The generation of an HIV-1 vaccine able to induce long-lasting protective immunity remains a main challenge. Here, we aimed to modify next-generation soluble, prefusion-stabilized, close-to-native, glycan-engineered clade C gp140 envelope (Env) trimers (sC23v4 KIKO and ConCv5 KIKO) for optimal display on the cell surface following homologous or heterologous vector delivery. Methods: A combination of the following modifications scored best regarding the preservation of closed, native-like Env trimer conformation and antigenicity when using a panel of selected broadly neutralizing (bnAb) and non-neutralizing (nnAb) monoclonal antibodies for flow cytometry: i) replacing the natural cleavage site with a native flexible linker and introducing a single amino acid substitution to prevent CD4 binding (*), ii) fusing a heterologous VSV-G-derived transmembrane moiety to the gp140 C-terminus, and iii) deleting six residues proximal to the membrane. Results: When delivering membrane-tethered sC23v4 KIKO* and ConCv5 KIKO* via DNA, VSV-GP, and NYVAC vectors, the two native-like Env trimers provide differential antigenicity profiles. Whereas such patterns were largely consistent among the different vectors for either Env trimer, the membrane-tethered ConCv5 KIKO* trimer adopted a more closed and native-like structure than sC23v4 KIKO*. In immunized mice, VSV-GP and NYVAC vectors expressing the membrane-tethered ConCv5 KIKO* administered in prime/boost combination were the most effective regimens for the priming of Env-specific CD4 T cells among all tested combinations. The subsequent booster administration of trimeric ConCv5 KIKO* Env protein preserved the T cell activation levels between groups. The evaluation of the HIV-1-specific humoral responses induced in the different immunization groups after protein boosts showed that the various prime/boost protocols elicited broad and potent antibody responses, preferentially of a Th1-associated IgG2a subclass, and that the obtained antibody levels remained high at the memory phase. Discussion: In summary, we provide a feasible strategy to display multiple copies of native-like Env trimers on the cell surface, which translates into efficient priming of sustained CD4+ T cell responses after vector delivery as well as broad, potent, and sustained antibody responses following booster immunizations with the homologous, prefusion-stabilized, close-to-native ConCv5 KIKO* gp140 Env trimer.

Antiviral Potential of Azelastine against Major Respiratory Viruses.

The Coronavirus Disease 2019 (COVID-19) pandemic and the subsequent increase in respiratory viral infections highlight the need for broad-spectrum antivirals to enable a quick and efficient reaction to current and emerging viral outbreaks. We previously demonstrated that the antihistamine azelastine hydrochloride (azelastine-HCl) exhibited in vitro antiviral activity against SARS-CoV-2. Furthermore, in a phase 2 clinical study, a commercial azelastine-containing nasal spray significantly reduced the viral load in SARS-CoV-2-infected individuals. Here, we evaluate the efficacy of azelastine-HCl against additional human coronaviruses, including the SARS-CoV-2 omicron variant and a seasonal human coronavirus, 229E, through in vitro infection assays, with azelastine showing a comparable potency against both. Furthermore, we determined that azelastine-HCl also inhibits the replication of Respiratory syncytial virus A (RSV A) in both prophylactic and therapeutic settings. In a human 3D nasal tissue model (MucilAirTM-Pool, Epithelix), azelastine-HCl protected tissue integrity and function from the effects of infection with influenza A H1N1 and resulted in a reduced viral load soon after infection. Our results suggest that azelastine-HCl has a broad antiviral effect and can be considered a safe option against the most common respiratory viruses to prevent or treat such infections locally in the form of a nasal spray that is commonly available globally.

Comparative Analysis of SARS-CoV-2 Antigenicity across Assays and in Human and Animal Model Sera.

The antigenic evolution of SARS-CoV-2 requires ongoing monitoring to judge the immune escape of newly arising variants. A surveillance system necessitates an understanding of differences in neutralization titers measured in different assays and using human and animal sera. We compared 18 datasets generated using human, hamster, and mouse sera, and six different neutralization assays. Titer magnitude was lowest in human, intermediate in hamster, and highest in mouse sera. Fold change, immunodominance patterns and antigenic maps were similar among sera. Most assays yielded similar results, except for differences in fold change in cytopathic effect assays. Not enough data was available for conclusively judging mouse sera, but hamster sera were a consistent surrogate for human first-infection sera.

Retained avidity despite reduced cross-binding and cross-neutralizing antibody levels to Omicron after SARS-COV-2 wild-type infection or mRNA double vaccination.

Introduction: The rapid evolution of SARS-CoV-2 has posed a challenge to long-lasting immunity against the novel virus. Apart from neutralizing function, binding antibodies induced by vaccination or infection play an important role in containing the infection. Methods: To determine the proportion of wild-type (WT)-generated antibodies recognizant of more recent variants, plasma samples from either SARS-CoV-2 WT-infected (n = 336) or double-mRNA (Comirnaty)-vaccinated individuals (n = 354, age and sex matched to the convalescent group) were analyzed for binding antibody capacity against the S1 protein of the BA.1 omicron variant. Results: Overall, 38.59% (95% CI, 37.01- 40.20) of WT-generated antibodies recognized Omicron BA.1 S1 protein [28.83% (95% CI, 26.73-30.91) after infection and 43.46% (95% CI, 41.61-45.31) after vaccination; p < 0.001]. Although the proportion of WT-generated binding and neutralizing antibodies also binding to BA.1 is substantially reduced, the avidity of the remaining antibodies against the Omicron variant was non-inferior to that of the ancestral virus: Omicron: 39.7% (95% CI: 38.1-41.3) as compared to the avidity to WT: 27.0% (95% CI, 25.5-28.4), respectively (p < 0.001). Furthermore, we noticed a modestly yet statistically significant higher avidity toward the Omicron epitopes among the vaccinated group (42.2%; 95% CI, 40.51-43.94) as compared to the convalescent counterparts (36.4%; 95% CI, 33.42-38.76) (p = 0.003), even after adjusting for antibody concentration. Discussion: Our results suggest that an aspect of functional immunity against the novel strain was considerably retained after WT contact, speculatively counteracting the impact of immune evasion toward neutralization of the strain. Higher antibody levels and cross-binding capacity among vaccinated individuals suggest an advantage of repeated exposure in generating robust immunity.

Characterizing SARS-CoV-2 neutralization profiles after bivalent boosting using antigenic cartography.

Since emergence of the initial SARS-CoV-2 BA.1, BA.2 and BA.5 variants, Omicron has diversified substantially. Antigenic characterization of these new variants is important to analyze their potential immune escape from population immunity and implications for future vaccine composition. Here, we describe an antigenic map based on human single-exposure sera and live-virus isolates that includes a broad selection of recently emerged Omicron variants such as BA.2.75, BF.7, BQ, XBB and XBF variants. Recent Omicron variants clustered around BA.1 and BA.5 with some variants further extending the antigenic space. Based on this antigenic map we constructed antibody landscapes to describe neutralization profiles after booster immunization with bivalent mRNA vaccines based on ancestral virus and either BA.1 or BA.4/5. Immune escape of BA.2.75, BQ, XBB and XBF variants was also evident in bivalently boosted individuals, however, cross-neutralization was improved for those with hybrid immunity. Our results indicate that future vaccine updates are needed to induce cross-neutralizing antibodies against currently circulating variants.

Immune response after two doses of the BNT162b2 COVID-19 vaccine and risk of SARS-CoV-2 breakthrough infection in Tyrol, Austria: an open-label, observational phase 4 trial.

BACKGROUND: Correlates of protection could help to assess the extent to which a person is protected from SARS-CoV-2 infection after vaccination (so-called breakthrough infection). We aimed to clarify associations of antibody and T-cell responses after vaccination against COVID-19 with risk of a SARS-CoV-2 breakthrough infection and whether measurement of these responses enhances risk prediction. METHODS: We did an open-label, phase 4 trial in two community centres in the Schwaz district of the Federal State of Tyrol, Austria, before the emergence of the omicron (B.1.1.529) variant of SARS-CoV-2. We included individuals (aged ≥16 years) a mean of 35 days (range 27-43) after they had received a second dose of the BNT162b2 (Pfizer-BioNTech) COVID-19 vaccine. We quantified associations between immunological parameters and breakthrough infection and assessed whether information on these parameters improves risk discrimination. The study is registered with the European Union Drug Regulating Authorities Clinical Trials Database, 2021-002030-16. FINDINGS: 2760 individuals (1682 [60·9%] female, 1078 [39·1%] male, mean age 47·4 years [SD 14·5]) were enrolled into this study between May 15 and May 21, 2021, 712 (25·8%) of whom had a previous SARS-CoV-2 infection. Over a median follow-up of 5·9 months, 68 (2·5%) participants had a breakthrough infection. In models adjusted for age, sex, and previous infection, hazard ratios for breakthrough infection for having twice the immunological parameter level at baseline were 0·72 (95% CI 0·60-0·86) for anti-spike IgG, 0·80 (0·70-0·92) for neutralising antibodies in a surrogate virus neutralisation assay, 0·84 (0·58-1·21) for T-cell response after stimulation with a CD4 peptide pool, and 0·77 (0·54-1·08) for T-cell response after stimulation with a combined CD4 and CD8 peptide pool. For neutralising antibodies measured in a nested case-control sample using a pseudotyped virus neutralisation assay, the corresponding odds ratio was 0·78 (0·62-1·00). Among participants with previous infection, the corresponding hazard ratio was 0·73 (0·61-0·88) for anti-nucleocapsid Ig. Addition of anti-spike IgG information to a model containing information on age and sex improved the C-index by 0·085 (0·027-0·143). INTERPRETATION: In contrast to T-cell response, higher levels of binding and neutralising antibodies were associated with a reduced risk of breakthrough SARS-CoV-2 infection. The assessment of anti-spike IgG enhances the prediction of incident breakthrough SARS-CoV-2 infection and could therefore be a suitable correlate of protection in practice. Our phase 4 trial measured both humoral and cellular immunity and had a 6-month follow-up period; however, the longer-term protection against emerging variants of SARS-CoV-2 remains unclear. FUNDING: None.

Therapeutic Efficacy of a VSV-GP-based Human Papilloma Virus Vaccine in a Murine Cancer Model.

Human papilloma virus (HPV) infections are associated with almost all cervical cancers and to a lower extend also with anogenital or oropharyngeal cancers. HPV proteins expressed in HPV-associated tumors are attractive antigens for cancer vaccination strategies as self-tolerance, which is associated with most endogenous tumor-associated antigens, does not need to be overcome. In this study, we generated a live attenuated cancer vaccine based on the chimeric vesicular stomatitis virus VSV-GP, which has previously proven to be a potent vaccine vector and oncolytic virus. Genes at an earlier position in the genome more to the 3' end are expressed stronger compared to genes located further downstream. By inserting an HPV16-derived antigen cassette consisting of E2, E6 and E7 into VSV-GP either at first (HPVp1) or fifth (HPVp5) position in VSV-GP's genome we aimed to analyze the effect of vaccine antigen position and consequently expression level on viral fitness, immunogenicity, and anti-tumoral efficacy in a syngeneic mouse tumor model. HPVp1 expressed higher amounts of HPV antigens compared to HPVp5 in vitro but had a slightly delayed replication kinetic which overall translated into increased HPV-specific T cell responses upon vaccination of mice. Immunization with both vectors protected mice in prophylactic and in therapeutic TC-1 tumor models with HPVp1 being more effective in the prophylactic setting. Taken together, VSV-GP is a promising candidate as therapeutic HPV vaccine and first position of the vaccine antigen in a VSV-derived vector seems to be superior to fifth position.

No evidence for epitranscriptomic m5C modification of SARS-CoV-2, HIV and MLV viral RNA.

The addition of chemical groups to cellular RNA to modulate RNA fate and/or function is summarized under the term epitranscriptomic modification. More than 170 different modifications have been identified on cellular RNA, such as tRNA, rRNA and, to a lesser extent, on other RNA types. Recently, epitranscriptomic modification of viral RNA has received considerable attention as a possible additional mechanism regulating virus infection and replication. N6-methyladenosine (m6A) and C5-methylcytosine (m5C) have been most broadly studied in different RNA viruses. Various studies, however, reported varying results with regard to number and extent of the modification. Here we investigated the m5C methylome of SARS-CoV-2, and we reexamined reported m5C sites in HIV and MLV. Using a rigorous bisulfite-sequencing protocol and stringent data analysis, we found no evidence for the presence of m5C in these viruses. The data emphasize the necessity for optimizing experimental conditions and bioinformatic data analysis.

B and T Cell Responses to SARS-CoV-2 Vaccination in Kidney and Liver Transplant Recipients with and without Previous COVID-19.

(1) Background: Vulnerable populations including transplant recipients are jeopardised by COVID-19. Herein, we report on B and T cell responses among liver and kidney organ recipients at our centre. (2) Methods: 23 liver and 45 kidney (14 thereof combined kidney/pancreas) transplanted patients were vaccinated with two doses of BNT162b2 followed by a booster dose of mRNA-1273 in 28 non-responders 4 months thereafter. Anti-SARS-CoV-2-Ig was measured by specific ELISA and virus neutralisation assay; T cell responses were measured by a spike protein-specific IFN-γ release assay. (3) Results: Compared to controls, B and T cell responses were weak in transplant recipients, particularly in those without prior exposure to SARS-CoV-2. Within this group, only 15% after the first and 58.3% after the second vaccination achieved seroconversion. A total of 14 out of 28 vaccination non-responders achieved a seroconversion after a third dose. Vaccination side effects were more frequent in healthy controls. The use of mycophenolate was associated with reduced anti-SARS-CoV-2-Ig production. (4) Conclusions: Our data confirm that vaccination responses are insufficient after standard vaccination in liver and kidney transplant recipients and are affected to a variable degree by specific immunosuppressants, particularly mycophenolate. Monitoring vaccination success and re-vaccinating those who are unresponsive seems prudent to achieve sufficient titres. Overall, prospective large-scale, multinational, multicentre studies or high-quality meta-analyses will be needed to generate personalised vaccination strategies in order to achieve protective immunity in high-risk, hard-to-immunize populations.

BA.2 and BA.5 omicron differ immunologically from both BA.1 omicron and pre-omicron variants.

Several studies have shown that SARS-CoV-2 BA.1 omicron is an immune escape variant. Meanwhile, however, omicron BA.2 and BA.5 became dominant in many countries and replaced BA.1. As both have several mutations compared to BA.1, we analyzed whether BA.2 and BA.5 show further immune escape relative to BA.1. Here, we characterized neutralization profiles against the BA.2 and BA.5 omicron sub-variants in plasma samples from individuals with different history of exposures to infection/vaccination and found that unvaccinated individuals after a single exposure to BA.2 had limited cross-neutralizing antibodies to pre-omicron variants and to BA.1. Consequently, our antigenic map including all Variants of Concern and BA.1, BA.2 and BA.5 omicron sub-variants, showed that all omicron sub-variants are distinct to pre-omicron variants, but that the three omicron variants are also antigenically distinct from each other. The antibody landscapes illustrate that cross-neutralizing antibodies against the current antigenic space, as described in our maps, are generated only after three or more exposures to antigenically close variants but also after two exposures to antigenically distant variants. Here, we describe the antigenic space inhabited by the relevant SARS-CoV-2 variants, the understanding of which will have important implications for further vaccine strain adaptations.

miR-142 favors naïve B cell residence in peripheral lymph nodes.

B lymphocyte development proceeds through a well-ordered sequence of steps, leading to the formation of a sizeable mature B population recognizing a diversity of antigens. These latter cells are ultimately responsible for the production of antibodies upon immune challenges. The detection of threats to the organism is facilitated by the ability of naïve follicular B cells, the main subset of mature B cells in mice, to circulate between lymphoid tissues in search of their cognate antigens. miRNA-mediated fine-tuning of mRNA stability and translation participates in the optimal expression of genetic programs. This regulatory mechanism has been shown to contribute to B cell biology, although the role of individual miRNAs remains understudied. Here, we selectively inactivated the miR-142 locus in B cells. As a consequence, the mature B compartment was visibly perturbed, in agreement with work in miR-142 knockout mice. However, our strategy allowed us to identify roles for the miR-142 locus in B cell physiology obscured by the complexity of the immune phenotype in the null mutant mice. Thus, these miRNAs are necessary for the proper formation of the pre-B cell compartment during development. More remarkably, naïve follicular B cells demonstrated altered migratory properties upon conditional inactivation of the miR-142 locus. The latter mutant cells expressed reduced levels of the homing molecule CD62L. They also migrated more efficiently towards sphingosine-1-phosphate in vitro and displayed an increased abundance of the sphingosine-1-phosphate receptor 1, compatible with improved lymphocyte egress in vivo. In line with these observations, the ablation of the miR-142 locus in B cells caused a paucity of B cells in the lymph nodes. Mutant B cell accumulation in the latter tissues was also compromised upon transfer into a wild-type environment. These changes coincided with suboptimal levels of FOXO1, a positive regulator of CD62L transcription, in mutant B cells. Overall, our findings indicate contributions for the miR-142 locus in various aspects of the B cell life cycle. Notably, this locus appears to favor the establishment of the migratory behavior required for naïve follicular B cell patrolling activity.

High Prevalence of Undocumented SARS-CoV-2 Infections in the Pediatric Population of the Tyrolean District of Schwaz.

Complementing the adult seroprevalence data collected at the time of the rapid SARS-CoV-2 mass vaccination in the district of Schwaz in 2021, we set out to establish the seroprevalence of SARS-CoV-2 among the pediatric population of the district. A total of 369 children, mean age 9.9 (SD 3.4), participated in the study, answering a structured questionnaire on the history of SARS-CoV-2 infection, household contacts, symptoms and history of vaccination. We determined binding and neutralizing antibody levels using plasma samples provided. We estimated the overall prevalence of SARS-CoV-2 infection in the general pediatric population at the time of the study using the census data from Statistik Austria and daily reports of officially confirmed cases. Excluding study participants who reported a history of PCR-confirmed infection, the age-standardized seroprevalence of previously unknown SARS-CoV-2 infection among the general pediatric population of the district was 27% (95% CI: 26.1-27.8). Adding this to the officially documented cases, the true overall prevalence was 32.8% (95% CI: 31.9-33.6) in contrast to the officially documented 8.0% (95% CI: 7.5-8.5) by June 2021. This translated into a proportion of 75.7% (95% CI: 74.4-77.0) of cases being officially undocumented, suggesting a high extent of silent SARS-CoV-2 infections in the pediatric population and possibly silent transmission.

Regimen of Coronavirus Disease 2019 Vaccination Influences Extent and Kinetics of Antibody Avidity.

We investigated antibody titers and avidity after heterologous versus homologous coronavirus disease 2019 vaccination over 6 months after the second dose. We found a significantly higher avidity in regimens including at least 1 dose of the adenoviral vector vaccine ChAdOx1-S compared with 2 doses of the mRNA vaccine BNT162b2.